Synthetic peptide amides

ABSTRACT

The invention relates to synthetic peptide amide ligands of the kappa opioid receptor and particularly to agonists of the kappa opioid receptor that exhibit low P 450  CYP inhibition and low penetration into the brain. The synthetic peptide amides of the invention conform to the structure of formula I: 
     
       
         
         
             
             
         
       
     
     Pharmaceutical compositions containing these compounds are useful in the prophylaxis and treatment of pain and inflammation associated with a variety of diseases and conditions. Such treatable pain includes visceral pain, neuropathic pain and hyperalgesia. Inflammation associated with conditions such as IBD and IBS, ocular and otic inflammation, other disorders and conditions such as pruritis, edema, hyponatremia, hypokalemia, ileus, tussis and glaucoma are treatable or preventable with the pharmaceutical compositions of the invention.

RELATED APPLICATIONS

This application claims priority to U.S. provisional application Ser.No. 60/858,109, filed Nov. 10, 2006, and to U.S. provisional applicationSer. No. 60/928,550, filed May 10, 2007, both of which are expresslyincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to synthetic peptide amides incorporating D-aminoacids in the peptide chain and more particularly to such syntheticpeptide amides that are kappa opioid receptor agonists, and methods fortheir use as prophylactic and therapeutic agents.

BACKGROUND

Kappa opioid receptors have been suggested as targets for interventionfor treatment or prevention of a wide array of diseases and conditionsby administration of kappa opioid receptor agonists. See for example,Jolivalt et al., Diabetologia, 49(11):2775-85; Epub Aug. 19, 2006),describing efficacy of asimadoline, a kappa receptor agonist in rodentdiabetic neuropathy; and Bileviciute-Ljungar et al., Eur. J. Pharm.494:139-46 (2004) describing the efficacy of kappa agonist U-50,488 inthe rat chronic constriction injury (CCI) model of neuropathic pain andthe blocking of its effects by the opioid antagonist, naloxone. Theseobservations support the use of kappa opioid receptor agonists fortreatment of diabetic, viral and chemotherapy-induced neuropathic pain.The use of kappa receptor agonists for treatment or prevention ofvisceral pain including gynecological conditions such as dysmenorrhealcramps and endometriosis has also been reviewed. See for instance,Riviere, Br. J. Pharmacol. 141:1331-4 (2004).

Kappa opioid receptor agonists have also been proposed for the treatmentof pain, including hyperalgesia. Hyperalgesia is believed to be causedby changes in the milieu of the peripheral sensory terminal occursecondary to local tissue damage. Tissue damage (e.g., abrasions, burns)and inflammation can produce significant increases in the excitabilityof polymodal nociceptors (C fibers) and high threshold mechanoreceptors(Handwerker et al. (1991) Proceeding of the VIth World Congress on Pain,Bond et al., eds., Elsevier Science Publishers BV, pp. 59-70; Schaibleet al. (1993) Pain 55:5-54). This increased excitability and exaggeratedresponses of sensory afferents is believed to underlie hyperalgesia,where the pain response is the result of an exaggerated response to astimulus. The importance of the hyperalgesic state in the post-injurypain state has been repeatedly demonstrated and appears to account for amajor proportion of the post-injury/inflammatory pain state. See forexample, Woold et al. (1993) Anesthesia and Analgesia 77:362-79; Dubneret al. (1994) In, Textbook of Pain, Melzack et al., eds.,Churchill-Livingstone, London, pp. 225-242.

Kappa opioid receptors have been suggested as targets for the preventionand treatment of cardiovascular disease. See for example, Wu et al.“Cardioprotection of Preconditioning by Metabolic Inhibition in the RatVentricular Myocyte—Involvement of kappa Opioid Receptor” (1999)Circulation Res vol. 84: pp. 1388-1395. See also Yu et al.“Anti-Arrhythmic Effect of kappa Opioid Receptor Stimulation in thePerfused Rat Heart: Involvement of a cAMP-Dependent Pathway” (1999) JMol Cell Cardiol. vol. 31(10): pp. 1809-1819.

It has also been found that development or progression of these diseasesand conditions involving neurodegeneration or neuronal cell death can beprevented, or at least slowed, by treatment with kappa opioid receptoragonists. This improved outcome is believed to be due to neuroprotectionby the kappa opioid receptor agonists. See for instance, Kaushik et al.“Neuroprotection in Glaucoma” (2003) J. Postgraduate Medicine vol. 49(1): pp. 90-95.

The presence of kappa opioid receptors on immune cells (Bidlak et al.,(2000) Clin. Diag. Lab. Immunol. 7(5):719-723) has been implicated inthe inhibitory action of a kappa opioid receptor agonist, which has beenshown to suppress HIV-1 expression. See Peterson P K et al., BiochemPharmacol. 2001, 61(19):1145-51.

Walker, Adv. Exp. Med. Biol. 521:148-60 (2003) appraised theanti-inflammatory properties of kappa agonists for treatment ofosteoarthritis, rheumatoid arthritis, inflammatory bowel disease andeczema. Bileviciute-Ljungar et al., Rheumatology 45:295-302 (2006)describe the reduction of pain and degeneration in Freund'sadjuvant-induced arthritis by the kappa agonist U-50,488.

Wikstrom et al., J. Am. Soc. Nephrol. 16:3742-7 (2005) describes the useof the kappa agonist, TRK-820 for treatment of uremic and opiate-inducedpruritis, and Ko et al., J. Pharmacol. Exp. Ther. 305:173-9 (2003)describe the efficacy of U-50,488 in morphine-induced pruritis in themonkey.

Application of peripheral opioids including kappa agonists for treatmentof gastrointestinal diseases has also been extensively reviewed. See forexample, Lembo, Diges. Dis. 24:91-8 (2006) for a discussion of use ofopioids in treatment of digestive disorders, including irritable bowelsyndrome (IBS), ileus, and functional dyspepsia.

Ophthalmic disorders, including ocular inflammation and glaucoma havealso been shown to be addressable by kappa opioids. See Potter et al.,J. Pharmacol. Exp. Ther. 309:548-53 (2004), describing the role of thepotent kappa opioid receptor agonist, bremazocine, in reduction ofintraocular pressure and blocking of this effect by norbinaltorphimine(norBNI), the prototypical kappa opioid receptor antagonist; andDortch-Carnes et al., CNS Drug Rev. 11(2):195-212 (2005). U.S. Pat. No.6,191,126 to Gamache discloses the use of kappa opioid agonists to treatocular pain. Otic pain has also been shown to be treatable byadministration of kappa opioid agonists. See U.S. Pat. No. 6,174,878also to Gamache.

Kappa opioid agonists increase the renal excretion of water and decreaseurinary sodium excretion (i.e., produces a selective water diuresis,also referred to as aquaresis). Many, but not all, investigatorsattribute this effect to a suppression of vasopressin secretion from thepituitary. Studies comparing centrally acting and purportedlyperipherally selective kappa opioids have led to the conclusion thatkappa opioid receptors within the blood-brain barrier are responsiblefor mediating this effect. Other investigators have proposed to treathyponatremia with nociceptin peptides or charged peptide conjugates thatact peripherally at the nociceptin receptor, which is related to butdistinct from the kappa opioid receptor (D. R. Kapusta, Life Sci.,60:15-21, 1997) (U.S. Pat. No. 5,840,696). U.S. Pat Appl. 20060052284.

SUMMARY OF THE INVENTION

The present invention provides synthetic peptide amides having theformula of formula I below, and stereoisomers, mixtures ofstereoisomers, prodrugs, pharmaceutically acceptable salts, hydrates,solvates, acid salt hydrates, N-oxides and isomorphic crystalline formsof the synthetic peptide amides of formula I:

In formula I, Xaa₁ represents an N-terminal amino acid that can be anyof (A)(A′)D-Phe, (A)(A′)(α-Me)D-Phe, D-Tyr,D-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid, D-tert-leucine,D-neopentylglycine, D-phenylglycine, D-homophenylalanine, or β(E)D-Ala,wherein each (A) and each (A′) are phenyl ring substituentsindependently chosen from —H, —F, —Cl, —NO₂, —CH₃, —CF₃, —CN, and—CONH₂, and wherein each (E) is independently chosen from the followingsubstituents: cyclobutyl, cyclopentyl, cyclohexyl, pyridyl, thienyl andthiazolyl.

Xaa₂ is a second amino acid that can be any of (A)(A′)D-Phe,3,4-dichloro-D-Phe, (A)(A′)(α-Me)D-Phe, D-1-naphthylalanine,D-2-naphthylalanine, D-Tyr, (E)D-Ala or D-Trp; wherein (A), (A′) and (E)are each independently chosen from the substituents listed above foreach of (A), (A′) and (E).

Xaa₃ is a third amino acid that can be any of D-norleucine, D-Phe,(E)D-Ala, D-Leu, (α-Me)D-Leu, D-homoleucine, D-Val, or D-Met, wherein(E) is independently chosen from the substituents listed above for (E).

Xaa₄ is a fourth amino acid that can be any of (B)₂D-arginine,(B)₂D-norarginine, (B)₂D-homoarginine, ζ-(B)D-homolysine,D-2,3-diaminopropionic acid, ε-(B)D-Lys, ε-(B)₂-D-Lys,D-aminomethylphenylalanine, amidino-D-aminomethylphenylalanine,γ-(B)₂D-γ-diamino butyric acid, δ-(B)₂α-(B)D-Orn,D-2-amino-3(4-piperidyl)propionic acid,D-2-amino-3(2-aminopyrrolidyl)propionic acid,D-α-amino-β-amidinopropionic acid, α-amino-4-piperidineacetic acid,cis-α,4-diaminocyclohexane acetic acid,trans-α,4-diaminocyclo-hexaneacetic acid, cis-α-amino-4-methylaminocyclohexane acetic acid, trans-α-amino-4-methylaminocyclohexane aceticacid, α-amino-1-amidino-4-piperidine acetic acid,cis-α-amino-4-guanidinocyclohexane acetic acid, ortrans-α-amino-4-guanidino-cyclohexane acetic acid, wherein each (B) isindependently either —H or C₁-C₄ alkyl, and (B′) is either —H or (α-Me).

The linking moiety, W can be any of the following three alternatives:(i) null, provided that when W is null, Y is N and is bonded to theC-terminus of Xaa₄ to form an amide; (ii) —NH—(CH₂)_(b)— with b equal to0, 1, 2, 3, 4, 5, or 6; or (iii) —NH—(CH₂)_(c)—O— with c equal to 2, or3, provided that Y is C. In each of the foregoing alternatives, (ii) and(iii) the N atom of W is bonded to the C-terminus of Xaa₄ to form anamide.

The moiety

in formula I is an optionally substituted 4 to 8-membered heterocyclicring moiety wherein all ring heteroatoms in the ring moiety are N, andwherein Y and Z are each independently C or N. However, when thisheterocyclic ring moiety is a six, seven or eight-membered ring, Y and Zmust be separated by at least two ring atoms. Further, when thisheterocyclic ring moiety has a single ring heteroatom which is N, thenthe ring moiety is non-aromatic.

The moiety V in formula I is C₁-C₆ alkyl. The operator, e is zero or 1,such that when e is zero, then V is null and R₁ and R₂ are directlybonded to the same or different ring atoms.

In the first of four alternative embodiments, the moiety R₁ in formula Ican be any of the following groups: —H, —OH, halo, —CF₃, —NH₂, —COOH,C₁-C₆ alkyl, amidino, C₁-C₆ alkyl-substituted amidino, aryl, optionallysubstituted heterocyclyl, Pro-amide, Pro, Gly, Ala, Val, Leu, Ile, Lys,Arg, Orn, Ser, Thr, CN, CONH₂, COR′, SO₂R′, CONR′R″, NHCOR′, OR′, orSO₂NR′R″; wherein the optionally substituted heterocyclyl is optionallysingly or doubly substituted with substituents independently chosen fromC₁-C₆ alkyl, C₁-C₆ alkoxy, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH,and amidino. The moieties R′ and R″ are each independently H, C₁-C₈alkyl, aryl, or heterocyclyl. Alternatively, R′ and R″ can be combinedto form a 4- to 8-membered ring, which ring is optionally substitutedsingly or doubly with substituents independently chosen from C₁-C₆alkyl, C₁-C₆ alkoxy, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH and amidino.The moiety R₂ can be any of —H, amidino, singly or doubly C₁-C₆alkyl-substituted amidino, —CN, —CONH₂, —CONR′R″, —NHCOR′, —SO₂NR′R″, or—COOH.

In a second alternative embodiment, the moieties R₁ and R₂ takentogether can form an optionally substituted 4- to 9-memberedheterocyclic monocyclic or bicyclic ring moiety which is bonded to asingle ring atom of the Y and Z-containing ring moiety.

In a third alternative embodiment, the moieties R₁ and R₂ taken togetherwith a single ring atom of the Y and Z-containing ring moiety can forman optionally substituted 4- to 8-membered heterocyclic ring moiety toform a spiro structure.

In a fourth alternative embodiment, the moieties R₁ and R₂ takentogether with two or more adjacent ring atoms of the Y and Z-containingring moiety can form an optionally substituted 4- to 9-memberedheterocyclic monocyclic or bicyclic ring moiety fused to the Y andZ-containing ring moiety.

In formula I, each of the optionally substituted 4-, 5-, 6-, 7-, 8- and9-membered heterocyclic ring moieties comprising R₁ and R₂ can be singlyor doubly substituted with substituents independently chosen from C₁-C₆alkyl, C₁-C₆ alkoxy, optionally substituted phenyl, oxo, —OH, —Cl, —F,—NH₂, —NO₂, —CN, —COOH and amidino. Further, when the Y and Z-containingring moiety of formula I is a six or seven-membered ring that has asingle ring heteroatom, and e is zero, then R₁ cannot be —OH, and R₁ andR₂ are not both —H.

When the Y and Z-containing ring moiety in formula I is a six-memberedring in which there are two ring heteroatoms wherein both Y and Z are N,and W is null, then the moiety —(V)_(c)R₁R₂ is attached to a ring atomother than Z. Moreover, under the foregoing conditions, if e is zero,then R₁ and R₂ cannot both be —H. Lastly, when Xaa₃ is D-Nle, then Xaa₄cannot be (B)₂D-Arg; and when Xaa₃ is D-Leu or (αMe)D-Leu, then Xaa₄cannot be δ-(B)₂α-(B′)D-Orn.

The invention also provides a selective kappa opioid receptor agonist(interchangeably referred to herein as a kappa receptor agonist orsimply as a kappa agonist) which is a synthetic peptide amide of theinvention, as described above.

The invention also provides a pharmaceutical composition, which includesa synthetic peptide amide of the invention and a pharmaceuticallyacceptable diluent, excipient or carrier.

Also provided is a method of treating or preventing a kappa opioidreceptor-associated disease or condition in a mammal. The methodincludes administering to the mammal a composition that includes aneffective amount of a synthetic peptide amide of the invention. Theinvention also provides uses of the synthetic peptide amides of theinvention for the preparation of medicaments and pharmaceuticalcompositions useful for the treatment of a kappa opioidreceptor-associated disease or condition in a mammal.

The invention further provides a method of treating or preventing akappa opioid receptor-associated disease or condition in a mammal,wherein a synthetic peptide amide of the invention is co-administeredwith a reduced dose of a mu opioid agonist analgesic compound to producea therapeutic analgesic effect, the mu opioid agonist analgesic compoundhaving an associated side effect, (especially respiratory depression,sedation, euphoria, antidiuresis, nausea, vomiting, constipation, andphysical tolerance, dependence, and addiction). The reduced dose of themu opioid agonist analgesic compound administered by this method haslower associated side effects than the side effects associated with thedose of the mu opioid agonist analgesic compound necessary to achievethe same therapeutic analgesic effect when administered alone.

The invention also provides a method of treating or preventingperipheral hyperalgesia, wherein the method includes topically applyingor locally administering to a mammal in need of the treatment, aneffective amount of a composition that includes ananti-hyperalgesically-effective amount of a synthetic peptide amide ofthe invention in a vehicle formulated for topical application or localadministration.

The invention also provides a method of treating or preventinghyponatremia or hypokalemia, and thereby treating or preventing adisease or condition associated with hyponatremia or hypokalemia, suchas congestive heart failure, liver cirrhosis, nephrotic syndrome,hypertension, or edema, and preferably where increased vasopressinsecretion is associated with said disease or disorder, wherein themethod includes administering to a mammal an aquaretically effectiveamount of a synthetic peptide amide of the invention in apharmaceutically acceptable diluent, excipient or carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Shows the general scheme used in the synthesis of compounds (1),(6), (7), (10) and (11). Steps a-s were carried out with the followingreactants or conditions: a) homopiperazine, DCM; b) Fmoc-D-Dap(ivDde)-OHor Fmoc-D-Dab(ivDde)-OH or Fmoc-D-Orn(Aloc)-OH or Fmoc-D-Orn(Cbz)-OH orFmoc-D-Lys(Dde)-OH or Fmoc-D-Arg(Pbf)-OH, DIC, HOBt, DMF; c) 25%piperidine in DMF; d) Fmoc-D-Leu-OH or Fmoc-D-Nle-OH, DIC, HOBt, DMF; e)Fmoc-D-Phe-OH, DIC, HOBt, DMF; f) Cbz-D-Phe-OH, DIC, HOBt, DMF; g) 4%hydrazine in DMF; h) Pd(PPh₃)₄, CHCl₃/AcOH/NMM; i) O—NBS—Cl, collidine,NMP; j) dimethyl-sulfate, DBU, NMR; k) mercaptoethanol, DBU, NMP; l)Cbz-OSu, DMF; m) acetone, AcOH, NaBH(OAc)₃, TMOF; n)1H-pyrazole-1-carboxamidine, DIEA, DMF; o) 50% TFA/DCM; p)S-Methyl-N-methylisothio-urea hydroiodide, DIEA, DMF; q)2-methylthio-2-imidazoline hydroiodide, DIEA, DMF; r) iodoethane, DIEA,DMF; s) TMSOTf/TFA/m-cresol.

FIG. 2: General scheme used in the synthesis of compounds (2)-(5), (8),(9) and (12)-(14). Steps a-k were carried out with the followingreactants or conditions: a) N-(1-Fmoc-piperidin-4-yl)-L-proline, DIEA,DCM; b) 25% piperidine/DMF; c) Fmoc-D-Lys(Dde)-OH, DIC, HOBt, DMF; d)Fmoc-D-Leu-OH, DIC, HOBt, DMF; e) Fmoc-D-Phe-OH, DIC, HOBt, DMF; f)Boc-D-Phe-OH, DIC, HOBt, DMF; g) 4% hydrazine in DMF; h) o-NBS—Cl,collidine, NMP; i) dimethylsulfate, DBU, NMP; j) mercaptoethanol, DBU,NMP; k) TFA/TIS/H₂O.

FIG. 3: General scheme used in the synthesis of compounds (15)-(24).Steps a-n were carried out with the following reactants or conditions:a) 35% piperidine, DMF; b) 1-Boc-4-N-Fmoc-amino-piperidine4-carboxylicacid, PyBOP, DIEA, DMF; c) (i) 35% piperidine, DMF; (ii) O—NBS—Cl,collidine, NMP; d) 30% TFA in DCM; e) Boc-D-Dap(Fmoc)-OH orBoc-D-Dab(Fmoc)-OH or Boc-D-Orn(Fmoc)-OH, PyBOP, DIEA, DMF; f)Boc-D-Leu-OH, PyBOP, DIEA, DMF; g) Boc-D-Phe-OH, PyBOP, DIEA, DMF; h)Boc-D-Phe-OH, PyBOP, DIEA, DMF; i) 2% DBU/DMF; j)1H-pyrazole-1-carboxamidine, DIEA, DMF; k) (i) acetone, TMOF, (ii)NaBH(OAc)₃, DMF; l) mercaptoethanol, DBU, NMP; m) Cu(OAc)₂, pyridine,DBU, DMF/H₂O; n) 95% TFA/H₂O.

FIG. 4: General scheme used in the synthesis of compounds (25)-(37).Steps a-h were carried out with the following reactants or conditions:a) EDCI, HOBt, DIEA, THF; b) TFA, DCM; c) Boc-D-Phe-OH, EDCI, HOBt,DIEA; d) H₂, Pd/C; e) D-Lys(Boc)-OAll, TBTU, DIEA, DMF; f) Pd(PPh₃)₄,pyrrolidine; g) HNRaRb, HBTU; h) HCl, dioxane.

FIG. 5: Concentration detected in plasma and brain of rats afteradministration of 3 mg/kg compound (2) over a 5 minute infusion periodthrough a jugular vein catheter. Concentration of compound (2) in ng/ml:open circles: plasma, solid circles: brain.

FIG. 6: Plasma concentrations of compound (6) after subcutaneousadministration of a single bolus of 1 mg/kg of the compound to ICR mice.Plasma was sampled at 5, 10, 15, 20, 30 60, 90 120, and 180 minutespost-injection.

FIG. 7: Plasma concentrations of compound (3) after intravenousadministration of a single bolus of 0.56 mg/kg of the compound tocynomolgus monkeys. Plasma was sampled at 2, 5, 10, 15, 30, 60, 120, and240 minutes post injection.

FIG. 8: Dose-response curves for compound (3) in ICR mice in the aceticacid-induced writhing assay (solid circles) and in the locomotion assay(solid squares).

FIG. 9: Dose response of compound (2)-mediated suppression of aceticacid-induced writhing in mice when delivered by the intravenous route.

FIG. 10: Effects of compound (2) on mechanical hypersensitivity inducedby L5/L6 spinal nerve ligation in rats. Open circles—vehicle alone;Solid circles—compound (2) at 0.1 mg/kg; open squares—compound (2) at0.3 mg/kg; solid squares—compound (2) at 1.0 mg/kg. ** denotes p<0.01;*** denotes p<0.001 vs. Vehicle (2 Way ANOVA, Bonferroni).

FIG. 11: Effect of Compound (2) at different concentrations onpancreatitis-induced abdominal hypersensitivity in rats. Dibutylindichloride or vehicle alone was administered intravenously andhypersensitivity assessed by abdominal probing with a von Frey filamentat 30 minute intervals. Hypersensitivity is expressed as number ofwithdrawals from ten probings. Open circles—vehicle alone; solidcircles—compound (2) at 0.1 mg/kg; open squares—compound (2) at 0.3mg/kg; solid squares—compound (2) at 1.0 mg/kg. ** denotes p<0.01; ***denotes p<0.001 vs. Vehicle (2 Way ANOVA, Bonferroni).

FIG. 12: Blocking of the effect of compound (2) on pancreatitis-inducedabdominal hypersensitivity by nor-BNI and naloxone methiodide in rats.Open column—vehicle alone, solid column—compound (2) at 1 mg/kg withnaloxone methiodide or norBNI as indicated. *** denotes p<0.001 vs.Vehicle+Vehicle (2 Way ANOVA, Bonferroni).

DETAILED DESCRIPTION

As used throughout this specification, the term “synthetic peptideamide” means a compound of the invention conforming to formula I, or astereoisomer, mixture of stereoisomers, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid salt hydrate, N-oxide orisomorphic crystalline form thereof. Where Xaa₁, Xaa₂, Xaa₃, and Xaa₄specify D amino acids in compounds of the invention, stereoisomers of acompound of the invention conforming to formula I are limited to thosecompounds having amino acids in the D configuration where so specifiedin Formula I. Stereoisomers of the invention do include compounds havingeither D or L configuration at chiral centers other than the alphacarbons of the four amino acids Xaa₁, Xaa₂, Xaa₃, and Xaa₄. Mixtures ofstereoisomers refer to mixtures of such stereoisomers of the invention.As used herein racemates refers to mixtures of stereoisomers havingequal proportions of compounds with D and L configuration at one or moreof the chiral centers other than the alpha carbons of Xaa₁, Xaa₂, Xaa₃,and Xaa₄ without varying the chirality of the alpha carbons of Xaa₁,Xaa₂, Xaa₃, and Xaa₄.

The nomenclature used to define peptides herein is specified by Schroder& Lubke, The Peptides, Academic Press, 1965, wherein, in accordance withconventional representation, the N-terminus appears to the left and theC-terminus to the right. Where an amino acid residue has isomeric forms,both the L-isomer form and the D-isomer form of the amino acid areintended to be covered unless otherwise indicated. Amino acids arecommonly identified herein by the standard three-letter code. TheD-isomer of an amino acid is specified by the prefix “D-” as in “D-Phe”which represents D-phenylalanine, the D-isomer of phenylalanine.Similarly, the L-isomer is specified by the prefix “L-” as in “L-Phe.”Peptides are represented herein according to the usual convention asamino acid sequences from left to right: N-terminus to C-terminus,unless otherwise specified.

As used herein, D-Arg represents D-arginine, D-Har representsD-homoarginine, which has a side chain one methylene group longer thanD-Arg, and D-Nar represents D-norarginine, which has a side chain onemethylene group shorter than D-Arg. Similarly, D-Leu means D-leucine,D-Nle means D-norleucine, and D-Hle represents D-homoleucine. D-Alameans D-alanine, D-Tyr means D-tyrosine, D-Trp means D-tryptophan, andD-Tic means D-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid. D-Valmeans D-valine and D-Met means D-methionine. D-Pro means D-proline,Pro-amide means the D- or L-form of proline amide. D-Pro amiderepresents D-proline with an amide formed at its carboxy moiety whereinthe amide nitrogen may be alkyl substituted, as in —NR_(a)R_(b), whereinR_(a) and R_(b) are each independently a C₁-C₆ alkyl group, or one ofR_(a) and R_(b) is —H. Gly means glycine, D-Ile means D-isoleucine,D-Ser means D-serine, and D-Thr means D-threonine. (E)D-Ala means theD-isomer of alanine which is substituted by the substituent (E) on theβ-carbon. Examples of such substituent (E) groups include cyclobutyl,cyclopentyl, cyclohexyl, pyridyl, thienyl and thiazoyl. Thus,cyclopentyl-D-Ala means the D-isomer of alanine which is substituted bycyclopentyl on the β-carbon. Similarly, D-Ala(2-thienyl) and(2-thienyl)D-Ala are interchangeable and both mean the D-isomer ofalanine substituted at the β-carbon with thienyl that is attached at the2-ring position.

As used herein, D-Nal means the D-isomer of alanine substituted bynaphthyl on the β-carbon. D-2Nal means naphthyl substituted D-alaninewherein the attachment to naphthalene is at the 2-position on the ringstructure and D-1Nal means naphthyl-substituted D-alanine wherein theattachment to naphthalene is at the 1-position on the ring structure. By(A)(A′)D-Phe is meant D-phenylalanine substituted on the phenyl ringwith one or two substituents independently chosen from halo, nitro,methyl, halomethyl (such as, for example, trifluoromethyl),perhalomethyl, cyano and carboxamide. By D-(4-F)Phe is meantD-phenylalanine which is fluoro-substituted in the 4-position of thephenyl ring. By D-(2-F)Phe is meant D-phenylalanine which isfluoro-substituted in the 2-position of the phenyl ring. By D-(4-Cl)Pheis meant D-phenylalanine which is chloro substituted in the 4-phenylring position. By (α-Me)D-Phe is meant D-phenylalanine which is methylsubstituted at the alpha carbon. By (α-Me)D-Leu is meant D-leucine whichis methyl substituted at the alpha carbon.

The designations (B)₂D-Arg, (B)₂D-Nar, and (B)₂D-Har representD-arginine, and D-norarginine D-homoarginine, respectively, each havingtwo substituent (B) groups on the side chain. D-Lys means D-lysine andD-Hlys means D-homolysine. ζ-(B)D-Hlys, ε-(B)D-Lys, and ε-(B)₂-D-Lysrepresent D-homolysine and D-lysine each having the side chain aminogroup substituted with one or two substituent (B) groups, as indicated.D-Orn means D-ornithine and δ-(B)α-(B′)D-Orn means D-ornithinesubstituted with (B′) at the alpha carbon and substituted with (B) atthe side chain δ-amino group.

D-Dap means D-2,3-diaminopropionic acid. D-Dbu represents the D-isomerof alpha, gamma-diamino butyric acid and (B)₂D-Dbu represents alpha,gamma-diamino butyric acid which is substituted with two substituent (B)groups at the gamma amino group. Unless otherwise stated, each of the(B) groups of such doubly substituted residues are independently chosenfrom H— and C₁-C₄-alkyl. As used herein, D-Amf means D-(NH₂CH₂-)Phe,i.e., the D-isomer of phenylalanine substituted with aminomethyl on itsphenyl ring and D-4Amf represents the particular D-Amf in which theaminomethyl is attached at the 4-position of the ring. D-Gmf meansD-Amf(amidino) which represents D-Phe wherein the phenyl ring issubstituted with —CH₂NHC(NH)NH₂. Amd represents amidino, —C(NH)NH₂, andthe designations (Amd)D-Amf and D-Amf(Amd) are also interchangeably usedfor D-Gmf. The designations fly and Ior are respectively used to meanisopropyl Lys and isopropyl Orn, wherein the side chain amino group isalkylated with an isopropyl group.

Alkyl means an alkane radical which can be a straight, branched, andcyclic alkyl group such as, but not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, t-butyl, sec-butyl, pentyl, cyclopentyl,hexyl, cyclohexyl, cyclohexylethyl. C₁ to C₈ alkyl refers to alkylgroups having between one and eight carbon atoms. Similarly, C₁-C₆ alkylrefers to alkyl groups having between one and six carbon atoms.Likewise, C₁-C₄ alkyl refers to alkyl groups having between one and fourcarbon atoms. By lower alkyl is meant C₁-C₆ alkyl. Me, Et, Pr, Ipr, Bu,and Pn are interchangeably used to represent the common alkyl groups:methyl, ethyl, propyl, isopropyl, butyl, and pentyl, respectively.Although the linkage for an alkyl group is typically at one end of analkyl chain, the linkage may be elsewhere in the chain, e.g. 3-pentylwhich may also be referred to as ethylpropyl, or 1-ethylprop-1-yl.Alkyl-substituted, such as C₁ to C₆ alkyl-substituted amidino, indicatesthat the relevant moiety is substituted with one or more alkyl groups.

Where a specified moiety is null, the moiety is absent and if suchmoiety is indicated to be attached to two other moieties, such two othermoieties are connected by one covalent bond. Where a connecting moietyis shown herein as attached to a ring at any position on the ring, andattached to two other moieties, such as R₁ and R₂, in the case where theconnecting moiety is specified to be null, then the R₁ and R₂ moietiescan each be independently attached to any position on the ring.

The terms “heterocycle”, “heterocyclic ring” and “heterocyclyl” are usedinterchangeably herein and refer to a ring or ring moiety having atleast one non-carbon ring atom, also called a heteroatom, which may be anitrogen, a sulfur, or an oxygen atom. Where a ring is specified ashaving a certain number of members, the number defines the number ofring atoms without reference to any substituents or hydrogen atomsbonded to the ring atoms. Heterocycles, heterocyclic rings andheterocyclyl moieties can include multiple heteroatoms independentlyselected from nitrogen, sulfur, or oxygen atom in the ring. Rings may besubstituted at any available position. For example, but withoutlimitation, 6- and 7-membered rings are often substituted in the 4-ringposition and 5-membered rings are commonly substituted in the3-position, wherein the ring is attached to the peptide amide chain atthe 1-ring position.

The term ‘saturated’ refers to an absence of double or triple bonds andthe use of the term in connection with rings describes rings having nodouble or triple bonds within the ring, but does not preclude double ortriple bonds from being present in substituents attached to the ring.The term “non-aromatic” refers in the context of a particular ring to anabsence of aromaticity in that ring, but does not preclude the presenceof double bonds within the ring, including double bonds which are partof an aromatic ring fused to the ring in question. Nor is a ring atom ofa saturated heterocyclic ring moiety precluded from being double-bondedto a non-ring atom, such as for instance a ring sulfur atom beingdouble-bonded to an oxygen atom substituent. As used herein,heterocycles, heterocyclic rings and heterocyclyl moieties also includesaturated, partially unsaturated and heteroaromatic rings and fusedbicyclic ring structures unless otherwise specified. A heterocycle,heterocyclic ring or heterocyclyl moiety can be fused to a second ring,which can be a saturated, partially unsaturated, or aromatic ring, whichring can be a heterocycle or a carbocycle. Where indicated, twosubstituents can be optionally taken together to form an additionalring. Rings may be substituted at any available position. A heterocycle,heterocyclic ring and heterocyclyl moiety can, where indicted, beoptionally substituted at one or more ring positions with one or moreindependently selected substituents, such as for instance, C₁-C₆ alkyl,C₃-C₈ cycloalkyl, C₁-C₆ alkoxy, halo C₁-C₆ alkyl, optionally substitutedphenyl, aryl, heterocyclyl, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOHand amidino. Suitable optional substituents of the phenyl substituentinclude for instance, but without limitation, one or more groupsselected from C₁-C₃ alkyl, C₁-C₃ alkoxy, halo C₁-C₃ alkyl, oxo, —OH,—Cl, —F, —NH₂, —NO₂, —CN, —COOH and amidino.

D-Phe and substituted D-Phe are examples of a suitable amino acid forresidue Xaa₁ in Formula I. The phenyl ring can be substituted at any ofthe 2-, 3- and/or 4-positions. Particular examples of permittedsubstitutions include, for instance, chlorine or fluorine at the 2- or4-positions. Also the alpha-carbon atom may be methylated. Otherequivalent residues which represent conservative changes to D-Phe canalso be used. These include D-Ala(cyclopentyl), D-Ala(thienyl), D-Tyrand D-Tic. The residue at the second position, Xaa₂ can also be D-Phe orsubstituted D-Phe with such substitutions including a substituent on the4-position carbon of the phenyl ring, or on both the 3- and 4-positions.Alternatively, Xaa₂ can be D-Trp, D-Tyr or D-alanine substituted bynaphthyl. The third position residue, Xaa₃ can be any non-polar aminoacid residue, such as for instance, D-Nle, D-Leu, (α-Me)D-Leu, D-Hle,D-Met or D-Val. However, D-Ala(cyclopropyl, cyclobutyl, cyclopentyl orcyclohexyl) or D-Phe can also be used as Xaa₃. The fourth positionresidue Xaa₄ can be any positively charged amino acid residue, such asfor instance, D-Arg and D-Har, which can be optionally substituted withlower alkyl groups, such as one or two ethyl groups. Alternatively,D-Nar and any other equivalent residues can be used, such as, forinstance, D-Lys or D-Orn (either of which can be ω-amino groupalkylated, for example by methyl or isopropyl groups, or methylated atthe α-carbon group). Moreover, D-Dbu, D-4-Amf (which can be optionallysubstituted with amidino), and D-Hlys are also suitable amino acids atthis position.

Compounds of the invention contain one or more chiral centers, each ofwhich has two possible three-dimensional spatial arrangements(configurations) of the four substituents around the central carbonatom. These are known as stereoisomers, and more specifically asenantiomers (all chiral centers inverted) or diastereoisomers (two ormore chiral centers, at least one chiral center remaining the same). Ina specific embodiment of the invention, the amino acids which make upthe tetrapeptide backbone, Xaa₁Xaa₂Xaa₃Xaa₄ are specified to be D-aminoacids i.e., the opposite configuration to those generally found inmammals. Reference to stereoisomers of the synthetic peptide amides ofthe invention concerns chiral centers other than the alpha carbons ofthe D-amino acids which make up Xaa₁-Xaa₄. Thus, stereoisomers ofsynthetic peptide amides that are embodiments of the invention whereineach of Xaa₁-Xaa₄ are specified to be D-amino acids, do not includeL-amino acids or racemic mixtures of the amino acids at these positions.Similarly, reference to racemates herein concerns a center other thanthe alpha carbons of the D-amino acids which make up Xaa₁-Xaa₄. Chiralcenters in the synthetic peptide amides of the invention for which astereoisomer may take either the R or S configuration include chiralcenters in the moiety attached to the carboxy-terminus of Xaa₄, and alsochiral centers in any amino acid side chain substituents of Xaa₁-Xaa₄.

The synthetic peptide amides of the invention described herein (alsointerchangeably referred to as synthetic peptide amide compounds,compounds of the invention, compound (number), or simply “thecompounds”) can be used or prepared in alternate forms. For example,many amino-containing compounds can be used or prepared as an acid salt.Often such salts improve isolation and handling properties of thecompound. For example, depending on the reagents, reaction conditionsand the like, compounds such as the synthetic peptide amides describedherein can be used or prepared, for example, as the hydrochloride ortosylate salts. Isomorphic crystalline forms, all chiral and racemicforms, N-oxide, hydrates, solvates, and acid salt hydrates, are alsocontemplated to be within the scope of the present invention.

Certain acidic or basic synthetic peptide amides of the presentinvention may exist as zwitterions. All forms of these synthetic peptideamide compounds, including free acid, free base and zwitterions, arecontemplated to be within the scope of the present invention. It is wellknown in the art that compounds containing both amino and carboxylgroups often exist in equilibrium with their zwitterionic forms. Thus,for any compound described herein that contains, for example, both aminoand carboxyl groups, it will also be understood to include thecorresponding zwitterion.

In certain embodiments the synthetic peptide amides of the inventionconform to Formula I:

In such embodiments Xaa₁-Xaa₂-Xaa₃-Xaa₄ is a tetrapeptide moiety whereinXaa₁ is the amino acid at the amino terminal, which can be (A)(A′)D-Phe,(A)(A′)(α-Me)D-Phe, D-Tyr, D-Tic, D-tert-leucine, D-neopentylglycine,D-phenylglycine, D-homophenylalanine, or β-(E)D-Ala, wherein each (A)and each (A′) are phenyl ring substituents independently selected from—H, —F, —Cl, —NO₂, —CH₃, —CF₃, —CN, and —CONH₂, and (E) is selected fromany of cyclobutyl, cyclopentyl, cyclohexyl, thienyl, pyridyl, orthiazolyl. The second amino acid in the tetrapeptide chain, Xaa₂, can beany of (A)(A′)D-Phe, 3,4-dichloro-D-Phe, (A)(A′)(α-Me)D-Phe, D-1Nal,D-2Nal, D-Tyr, (E)D-Ala or D-Trp. The third amino acid in thetetrapeptide chain, Xaa₃ can be D-Nle, D-Phe, (E)D-Ala, D-Leu,(α-Me)D-Leu, D-Hle, D-Val, or D-Met. The fourth amino acid in thetetrapeptide chain, Xaa₄ can be any of the following: (B)₂D-Arg,(B)₂D-Nar, (B)₂D-Har, ζ-(B)D-Hlys, D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys,D-Amf, amidino-D-Amf, γ-(B)₂D-Dbu, δ-(B)₂α-(B′)D-Orn,D-2-amino-3(4-piperidyl)propionic acid,D-2-amino-3(2-amino-pyrrolidyl)-propionic acid,D-α-amino-β-amidinopropionic acid, α-amino-4-piperidine acetic acid,cis-α,4-diaminocyclohexane acetic acid, trans-α,4-diaminocyclohexaneacetic acid, cis-α-amino-4-methylamino-cyclohexane acetic acid,trans-α-amino-4-methylamino cyclohexane acetic acid,α-amino-1-amidino-4-piperidine acetic acid,cis-α-amino-4-guanidinocyclohexane acetic acid, ortrans-α-amino-4-guanidinocyclohexane acetic acid, wherein each (B) canbe separately chosen from —H and a C₁-C₄ alkyl, and (B′) can be —H or an(α-Me) group.

In certain embodiments of the invention, the optional linker group W isabsent (i.e., null), provided that in such case, Y is N. In otherembodiments, W is —N—(CH₂)_(b) with b equal to zero, 1, 2, 3, 4, 5, or6. In still other embodiments, W is —N—(CH₂)_(c)—O— with c equal to 2,or 3, provided that in such embodiments, Y is C.

In particular embodiments of the invention, the moiety

in formula I is an optionally substituted 4- to 8-membered heterocyclicring moiety, wherein all ring heteroatoms in the ring moiety are N, andwherein Y and Z are each independently C or N, and are not adjacent ringatoms. In such embodiments, when this heterocyclic ring moiety is a six,seven or eight-membered ring, the ring atoms Y and Z are separated by atleast two other ring atoms. In such embodiments, when this heterocyclicring moiety has a single ring heteroatom which is N, the ring moiety isnon-aromatic.

In certain particular embodiments of the invention, a connecting moiety,V is directly bonded to the Y- and Z-containing ring. V is a C₁-C₆ alkylwhich can be substituted with groups R₁ and R₂. The substituent R₁ canbe any of —H, —OH, halo, —CF₃, —NH₂, —COOH, C₁-C₆ alkyl, amidino, C₁-C₆alkyl-substituted amidino, aryl, optionally substituted heterocyclyl,Pro-amide, Pro, Gly, Ala, Val, Leu, Ile, Lys, Arg, Orn, Ser, Thr, —CN,—CONH₂, —COR′, —SO₂R′, —CONR′R″, —NHCOR′, OR′, or —SO₂NR′R″. Theoptionally substituted heterocyclyl can be singly or doubly substitutedwith substituents independently chosen from C₁-C₆ alkyl, C₁-C₆ alkoxy,oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino. The moieties R′and R″ are each independently H, C₁-C₈ alkyl, aryl, or heterocyclyl.Alternatively, R′ and R″ can be combined to form a 4- to 8-memberedring, which ring is optionally substituted singly or doubly withsubstituents independently chosen from C₁-C₆ alkyl, C₁-C₆ alkoxy, —OH,—Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino. The substituent R₂ can beany of —H, amidino, singly or doubly C₁-C₆ alkyl-substituted amidino,—CN, —CONH₂, —CONR′R″, —NHCOR′, —SO₂NR′R″, or —COOH.

In other particular embodiments, V is absent and the substituent groupsR₁ and R₂ are directly bonded to the same or different ring atoms of theY- and Z-containing heterocyclic ring.

In one alternative aspect of certain embodiments, the moieties R₁ and R₂taken together can form an optionally substituted 4- to 9-memberedheterocyclic monocyclic or bicyclic ring moiety which is bonded to asingle ring atom of the Y and Z-containing ring moiety. In oneparticular embodiment, the moieties R₁ and R₂ form an optionallysubstituted 4- to 9-membered heterocyclic monocyclic or bicyclic ringmoiety which is directly bonded to a single ring atom of the Y andZ-containing ring moiety.

In a second alternative aspect of certain embodiments, the moieties R₁and R₂ taken together with a single ring atom of the Y and Z-containingring moiety can form an optionally substituted 4- to 8-memberedheterocyclic ring moiety to form a spiro structure.

In a third alternative aspect of certain embodiments, the moieties R₁and R₂ taken together with two or more adjacent ring atoms of the Y andZ-containing ring moiety can form an optionally substituted 4- to9-membered heterocyclic monocyclic or bicyclic ring moiety fused to theY and Z-containing ring moiety.

In particular embodiments, each of the optionally substituted 4-, 5-,6-, 7-, 8- and 9-membered heterocyclic ring moieties comprising R₁ andR₂ can be singly or doubly substituted with substituents independentlychosen from C₁-C₆ alkyl, C₁-C₆ alkoxy, optionally substituted phenyl,oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, and amidino.

In certain particular embodiments, when the Y- and Z-containing ringmoiety is a six or seven-membered ring that includes a single ringheteroatom, and when one of Y and Z is C and the other of Y and Z is N,and e is zero, then R₁ cannot be —OH, and R₁ and R₂ are not both —H.Further, when the Y and Z-containing ring moiety is a six-membered ringthat includes two ring heteroatoms, wherein both Y and Z are N and W isnull, then the moiety —(V)_(c)R₁R₂ is attached to a ring atom other thanZ. Moreover, if e is zero, then R₁ and R₂ cannot both be —H. Lastly,when Xaa₃ is D-Nle, then Xaa₄ cannot be (B)₂D-Arg; and when Xaa₃ isD-Leu or (αMe)D-Leu, then Xaa₄ cannot be δ-(B)₂α-(B′)D-Orn.

In certain embodiments, the present invention also provides a syntheticpeptide amide having the formula:

or a stereoisomer, racemate, prodrug, pharmaceutically acceptable salt,hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystallineform thereof; wherein Xaa₁ is selected from the group consisting of(A)(A′)D-Phe, (α-Me)D-Phe, D-Tyr, D-Tic, D-phenylglycine,D-homophenylalanine, and β-(E)D-Ala, wherein (A) and (A′) are eachphenyl ring substituents independently selected from the groupconsisting of —H, —F, —Cl, —NO₂, —CH₃, —CF₃, —CN, and —CONH₂, andwherein (E) is selected from the group consisting of cyclobutyl,cyclopentyl, cyclohexyl, pyridyl, thienyl and thiazolyl; Xaa₂ isselected from the group consisting of (A)(A′)D-Phe, (α-Me)D-Phe, D-1Nal,D-2Nal, D-Tyr, (E)D-Ala and D-Trp; Xaa₃ is selected from the groupconsisting of D-Nle, D-Phe, (E)D-Ala, D-Leu, (α-Me)D-Leu, D-Hle, D-Val,and D-Met; Xaa₄ is selected from the group consisting of (B)₂D-Arg,(B)₂D-nArg, (B)₂D-Har, ζ-(B)D-Hlys, D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys,D-Amf, amidino-D-Amf, γ-(B)₂D-Dbu, δ-(B)₂α-(B′)D-Orn,D-2-amino-3(4-piperidyl)propionic acid,D-2-amino-3(2-aminopyrrolidyl)propionic acid,D-α-amino-β-amidinopropionic acid, (R)-α-amino-4-piperidineacetic acid,cis-α,4-diaminocyclohexane acetic acid,trans-α,4-diaminocyclohexaneacetic acid,cis-α-amino-4-methylaminocyclohexane acetic acid,trans-α-amino-4-methylaminocyclohexane acetic acid,α-amino-1-amidino-4-piperidineacetic acid,cis-α-amino-4-guanidinocyclohexane acetic acid, andtrans-α-amino-4-guanidinocyclohexane acetic acid, wherein each (B) isindependently selected from the group consisting of H and C₁-C₄ alkyl,and (B′) is H or (α-Me). The group W is selected from the groupconsisting of:Null, provided that when W is null, Y is N;—N—(CH₂)_(b) with b equal to zero, 1, 2, 3, 4, 5, or 6; and—N—(CH₂)_(c)—O— with c equal to 2, or 3, provided that Y is C;and the moiety

is an optionally substituted 4-8 membered saturated mono- or dinitrogenheterocyclic ring moiety, wherein no ring atom other than Y and Z is aheteroatom, Y is C or N, Z is C or N, and at least one of Y and Z is N,and provided that in the case of a 4 or 5 membered heterocyclic ring,either Y or Z is C, and in the case of a dinitrogen heterocycle, Y and Zare separated by two or more ring carbon atoms;V is C₁-C₆ alkyl, and e is zero or 1, wherein when e is zero, then V isnull and R₁ and R₂ are directly bonded to the same or different ringatoms;R₁ is H, OH, —NH₂, —COOH, C₁-C₆ alkyl, amidino, C₁-C₆ alkyl-substitutedamidino, dihydroimidazole, Pro-amide, Pro, Gly, Ala, Val, Leu, Ile, Lys,Arg, Orn, Ser, Thr, —CN, —CONH₂, —CONR′R″, —NHCOR′, —OR′, or —SO₂NR′R″,wherein R′ and R″ are each independently H, or C₁-C₈ alkyl, or R′ and R″are combined to form a 4- to 8-membered ring which ring is optionallysubstituted singly or doubly with substituents independently selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, —OH, —Cl, —F,—NH₂, —NO₂, —CN, and —COOH, amidino; and R₂ is H, amidino, singly ordoubly C₁-C₆ alkyl-substituted amidino, —CN, —CONH₂, —CONR′R″, —NHCOR′,—SO₂NR′R″, or —COOH; provided that when the Y and Z-containing ringmoiety is a six or seven membered ring and when one of Y and Z is C ande is zero, then R₁ is not OH, and R₁ and R₂ are not both H; providedthat when the Y and Z-containing ring moiety is a six membered ring,both Y and Z are N and W is null, then —(V)_(e)R₁R₂ is attached to aring atom other than Z; and if e is zero, then R₁ and R₂ are not both—H; and lastly, provided that when Xaa₃ is D-Nle, then Xaa₄ cannot be(B)₂D-Arg, and when Xaa₃ is D-Leu or (αMe)D-Leu, then Xaa₄ cannot beδ-(B)₂α-(B′)D-Orn.

In one embodiment, the present invention provides a synthetic peptideamide of formula I, wherein Xaa₁Xaa₂ is D-Phe-D-Phe, Xaa₃ is D-Leu orD-Nle and Xaa₄ is chosen from (B)₂D-Arg, D-Lys, (B)₂D-Har, ζ-(B)D-Hlys,D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf, γ-(B)₂D-Dbu andδ-(B)₂α-(B′)D-Orn. In a particular aspect of the above embodiment, Xaa₄is chosen from D-Lys, (B)₂D-Har, ε-(B)D-Lys, and ε-(B)₂-D-Lys.

In another embodiment, the invention provides a synthetic peptide amideof formula I, wherein W is null, Y is N and Z is C. In a particularaspect of the above embodiment, the Y and Z-containing ring moiety is asix-membered saturated ring comprising a single ring heteroatom.

In another embodiment, the invention provides a synthetic peptide amideof formula I, wherein Y and Z are both N and are the only ringheteroatoms in the Y and Z-containing ring moiety.

In another embodiment, when the Y- and Z-containing ring moiety is asaturated six-membered ring that includes two ring heteroatoms and W isnull, then Z is a carbon atom.

In yet another embodiment, the invention provides a synthetic peptideamide of formula I, wherein R₁ and R₂ taken together with zero, one ortwo ring atoms of the Y and Z-containing ring moiety comprise amonocyclic or bicyclic 4-9 membered heterocyclic ring moiety. In aparticular aspect of the above embodiment, R, and R₂ taken together withone ring atom of the Y and Z-containing ring moiety comprise a 4- to8-membered heterocyclic ring moiety which with the Y and Z-containingring moiety forms a spiro structure and W is null.

In still another embodiment, the invention provides a synthetic peptideamide of formula I, wherein e is zero and R₁ and R₂ are bonded directlyto the same ring carbon atom.

In a further embodiment, the invention provides a synthetic peptideamide of formula I, wherein R₁ is H, OH, —NH₂, —COOH, —CH₂COOH, C₁-C₃alkyl, amidino, C₁-C₃ alkyl-substituted amidino, dihydroimidazole,D-Pro, D-Pro amide, or —CONH₂ and wherein R₂ is H, —COOH, or C₁-C₃alkyl.

In another embodiment, the invention provides a synthetic peptide amideof formula I, wherein the moiety:

is selected from the group consisting of:

In still yet another embodiment, the invention provides a syntheticpeptide amide of formula I wherein the moiety:

is neither a proline moiety, nor a substituted proline moiety, nor is ita proline moiety wherein R₁ or R₂ contains an amide moiety.

In still yet another embodiment, the invention provides a syntheticpeptide amide of formula I, wherein it is provided that when W is null,the Y and Z-containing ring moiety is a saturated 5-membered ring withonly a single ring heteroatom, e is zero and either R₁ or R₂ is attachedto a ring carbon adjacent to Y, then R₁ is selected from the groupconsisting of —H, —OH, halo, —CF₃, —NH₂, C₁-C₆ alkyl, amidino, C₁-C₆alkyl-substituted amidino, aryl, Pro, Gly, Ala, Val, Leu, Ile, Lys, Arg,Orn, Ser, Thr, CN, —SO₂R′, —NHCOR′, —OR′ and —SO₂NR′R″ and R₂ isselected from the group consisting of —H, amidino, singly or doublyC₁-C₆ alkyl-substituted amidino, —CN, —NHCOR′, and —SO₂NR′R″.

In still yet another embodiment, the invention provides a syntheticpeptide amide of formula I having an EC₅₀ of less than about 500 nM fora kappa opioid receptor. In a particular aspect, the synthetic peptideamide can have an EC₅₀ of less than about 100 nM for a kappa opioidreceptor. In a more particular aspect, the synthetic peptide amide canhave an EC₅₀ of less than about 20 nM for a kappa opioid receptor. Inmost particular aspect, the synthetic peptide amide can have an EC₅₀ ofless than about 1 nM for a kappa opioid receptor. The compounds of theforegoing embodiment can have an EC₅₀ that is at least 10 times greaterfor a mu and a delta opioid receptor than for a kappa opioid receptor,preferably at least 100 times greater, and most preferably at least 1000times greater (e.g., an EC₅₀ of less than about 1 nM for a kappa opioidreceptor, and EC₅₀ values of greater than about 1000 nM for a mu opioidreceptor and a delta opioid receptor).

In still yet another embodiment, the invention provides a syntheticpeptide amide of formula I which at an effective concentration exhibitsno more than about 50% inhibition of any of P₄₅₀ CYP1A2, CYP2C9, CYP2C19or CYP 2D6 by the synthetic peptide amide at a concentration of 10 uMafter 60 minutes incubation with human liver microsomes.

In still a further embodiment, the invention provides a syntheticpeptide amide of formula I, which at a dose of about 3 mg/kg in ratreaches a peak plasma concentration of the synthetic peptide amide thatis at least about five fold higher than the peak concentration in brain.In one particular aspect of the above embodiment, the synthetic peptideamide has an ED₅₀ for a sedative effect in a locomotion-reduction assayin a mouse at least about ten times the ED₅₀ of the synthetic peptideamide for an analgesic effect in a writhing assay in a mouse.

In still a further embodiment, the invention provides a syntheticpeptide amide of formula I having at least about 50% of maximum efficacyat about 3 hours post administration of a dose of about 3 mg/kg of thesynthetic peptide amide in a rat.

In one embodiment, the invention provides a pharmaceutical compositionthat includes a synthetic peptide amide of formula I and apharmaceutically acceptable excipient or carrier.

In another embodiment, the invention provides a method of treating orpreventing a kappa opioid receptor-associated disease or condition in amammal; the method includes administering to the mammal a compositioncomprising an effective amount of a synthetic peptide amide according toformula I sufficient to treat or prevent the kappa opioidreceptor-associated disease or condition.

A variety of assays may be employed to test whether the syntheticpeptide amides of the invention exhibit high affinity and selectivityfor the kappa opioid receptor, long duration of in vivo bioactivity, andlack of CNS side effects. Receptor assays are well known in the art andkappa opioid receptors from several species have been cloned, as have muand delta opioid receptors. Kappa opioid receptors as well as mu anddelta opioid receptors are classical, seven transmembrane-spanning, Gprotein-coupled receptors. Although these cloned receptors readily allowa particular candidate compound, e.g., a peptide or peptide derivative,to be screened, natural sources of mammalian opioid receptors are alsouseful for screening, as is well known in the art (Dooley C T et al.Selective ligands for the mu, delta, and kappa opioid receptorsidentified from a single mixture based tetrapeptide positional scanningcombinatorial library. J. Biol. Chem. 273:18848-56, 1998). Thus,screening against both kappa and mu opioid receptors, whether ofrecombinant or natural origin, may be carried out in order to determinethe selectivity of the synthetic peptide amides of the invention for thekappa over the mu opioid receptor.

In a particular embodiment, the synthetic peptide amides of theinvention are selective kappa opioid receptor agonists. The potency ofthe synthetic peptide amides of the invention as agonists for aparticular receptor can be measured as a concentration at which halfmaximal effect is achieved expressed as an EC₅₀ value. Potency of thesynthetic peptide amides of the invention as kappa opioid agonists,expressed as the percent of maximal observable effect, can be determinedby a variety of methods well known in the art. See for example, Endoh Tet al., 1999, Potent Antinociceptive Effects of TRK-820, a Novelκ-Opioid Receptor Agonist, Life Sci. 65 (16) 1685-94; and Kumar V etal., Synthesis and Evaluation of Novel peripherally Restricted κ-OpioidReceptor Agonists, 2005 Bioorg Med Chem Letts 15: 1091-1095.

Examples of such assay techniques for determination of EC₅₀ values areprovided below. Many standard assay methods for characterization ofopioid ligands are well known to those of skill in the art. See, forexample, Waldhoer et al., (2004) Ann. Rev. Biochem. 73:953-990, andSatoh & Minami (1995) Pharmac. Ther. 68(3):343-364 and references citedtherein.

In certain particular embodiments, the synthetic peptide amides of theinvention are kappa opioid receptor agonists with an EC₅₀ of less thanabout 500 nM. In other embodiments, the synthetic peptide amides have anEC₅₀ of less than about 100 nM as kappa opioid receptor agonists. Instill other embodiments, the synthetic peptide amides have an EC₅₀ ofless than about 10 nM as kappa opioid receptor agonists. In particularembodiments the synthetic peptide amides of the invention have an EC₅₀of less than about 1.0 nM, less than about 0.1 nM, or less than about0.1 nM, or even less than about 0.01 nM as kappa opioid receptoragonists.

In particular embodiments, the synthetic peptide amides of the inventionare highly selective for kappa over mu opioid receptors. In certainembodiments the synthetic peptide amides of the invention have EC₅₀values for the mu opioid receptor that are at least about a hundredtimes higher than the corresponding EC₅₀ values for the kappa opioidreceptor. In particular embodiments, the synthetic peptide amides of theinvention have EC₅₀ values for the mu opioid receptor that are at leastabout a thousand times higher than the corresponding EC₅₀ values for thekappa opioid receptor. Alternatively, the selectivity of the syntheticpeptide amides of the invention can be expressed as a higher EC₅₀ for amu opioid receptor than for a kappa opioid receptor. Thus, in particularembodiments, the synthetic peptide amides of the invention have EC₅₀values of greater than about 10 uM for the mu opioid receptor and EC₅₀values of less than about 10 nM, and in other embodiments less thanabout 1.0 nM, or even less than about 0.01 nM for the kappa opioidreceptor. In another embodiment, the particular synthetic peptide amidecan have an EC₅₀ of less than about 1 nM for a kappa opioid receptor andan EC₅₀ of greater than about 1000 nM for a mu opioid receptor, or for adelta opioid receptor.

Another property of the synthetic peptide amides of the invention istheir characteristic property of low inhibition of the cytochrome P₄₅₀isozymes. The cytochrome P₄₅₀ isozymes constitute a large superfamily ofhaem-thiolate proteins responsible for metabolic oxidative inactivationof many therapeutics and other bioactive compounds. Usually, they act asterminal oxidases in multicomponent electron transfer chains, alsoreferred to as cytochrome P₄₅₀-containing monooxygenase systems.

Over fifty different cytochrome P₄₅₀ isozymes have been identified andhave been classified into families grouped by genetic relatedness asassessed by nucleic acid sequence homology. Most abundant among thecytochrome P₄₅₀ isozymes in human cells are the 1A2 and 3A4 isozymes,although isozymes 2B6, 2C9, 2C19, 2D6, and 2E1 also contributesignificantly to oxidative inactivation of administered therapeutics.While inhibition of the cytochrome P₄₅₀ isozymes may be useful inprolonging the time post in vivo administration during which aneffective concentration of the synthetic peptide amides of the inventionis maintained, it also prolongs the persistence of any co-administeredtherapeutic compound that is subject to oxidation by cytochrome P₄₅₀.This increase in persistence may cause the co-administered therapeuticto persist beyond the period that is optimal for therapy, or may causethe in vivo concentration to exceed the desired levels or safelytolerated levels. Such increases in persistence and/or increases inconcentration are difficult to accurately quantify and are preferablyavoided. Therapeutics that show little or no inhibition of the activityof the cytochrome P₄₅₀ isozymes do not have this potential problem andcan be more safely co-administered with other therapeutics without riskof affecting the rate of inactivation of the co-administered therapeuticcompound by the cytochrome P₄₅₀ isozymes.

Particular embodiments of the synthetic peptide amides of the inventionshow low inhibition of the cytochrome P₄₅₀ isozymes at therapeuticconcentrations of the synthetic peptide amides, while others showessentially no inhibition of the cytochrome P₄₅₀ isozymes at therapeuticconcentrations. In some embodiments, the synthetic peptide amides at aconcentration of 10 uM show less than about 50% inhibition of cytochromeP₄₅₀ isozymes CYP1A2, CYP2C9, CYP2C19 or CYP2D6. In particularembodiments, the synthetic peptide amides at a concentration of 10 uMshow less than about 20% inhibition of any of these cytochrome P₄₅₀isozymes. In very particular embodiments, the synthetic peptide amidesat a concentration of 10 uM show less than about 10% inhibition of anyof these cytochrome P₄₅₀ isozymes.

In another embodiment, the synthetic peptide amides of the invention atan effective concentration exhibit no more than about 50% inhibition ofany of P₄₅₀ CYP1A2, CYP2C9, CYP2C19 or CYP 2D6 by the synthetic peptideamide at a concentration of 10 uM after 60 minutes incubation with humanliver microsomes.

The synthetic peptide amides of the invention when administered to amammal or a human patient at a therapeutically effective concentrationexhibit low or essentially no penetration across the blood-brainbarrier. Kappa opioid receptors (hereinafter interchangeably referred toas kappa receptors) are distributed in peripheral tissues including theskin and somatic tissues, as well as the viscera in humans and othermammals. Kappa receptors are also found in the brain. Activation of thekappa receptors in peripheral tissues causes suppression of pain andinflammatory responses, while activation of the kappa receptors in thebrain causes sedative effects and may also lead to severe dysphoria andhallucinations. In certain embodiments, the synthetic peptide amide ofthe invention when administered at therapeutically effectiveconcentrations exhibit essentially no penetration across the blood-brainbarrier and therefore minimize or even completely obviate the sedative,hallucinogenic effects of many other kappa agonists that show somepenetration across the blood-brain barrier.

One useful measure of the extent to which the synthetic peptide amidesof the invention cross the blood-brain barrier is the ratio of the peakplasma concentration to the concentration in brain tissue. In particularembodiments, the synthetic peptide amides of the invention whenadministered at dose of about 3 mg/kg, exhibit at least about a fivefold lower concentration of the synthetic peptide amide in brain than inplasma at the time when peak plasma concentration is reached.

Another useful measure of the extent to which the synthetic peptideamides of the invention cross the blood-brain barrier is the ratio ofthe dose required to achieve a sedative effect and the dose required toachieve an analgesic effect. The analgesic and sedative effects of kappareceptor stimulation by kappa receptor agonists can be measured bystandard assays well known to those of skill in the art.

In particular embodiments, the synthetic peptide amides of the inventionhave an ED₅₀ for a sedative effect that is at least about ten times theED₅₀ for an analgesic effect. In particular embodiments, the syntheticpeptide amides of the invention have an ED₅₀ for a sedative effect thatis at least about thirty times the ED₅₀ for an analgesic effect. Instill other embodiments, the synthetic peptide amides of the inventionhave an ED₅₀ for a sedative effect that is at least about fifty timesthe ED₅₀ for an analgesic effect.

In another embodiment, the synthetic peptide amides of the inventionhave an ED₅₀ for a sedative effect in a locomotion-reduction assay in amouse at least about ten times the ED₅₀ of the synthetic peptide amidefor an analgesic effect in a writhing assay in a mouse.

Another useful predictor of the extent to which the synthetic peptideamides of the invention would be expected to cross the blood-brainbarrier is provided by the membrane permeability values of the syntheticpeptide amides into a human cell or other mammalian cell when deliveredat a therapeutically relevant concentration. In certain embodiments, thesynthetic peptide amides of the invention at therapeutically relevantconcentrations exhibit low or essentially no ability to penetrate amonolayer of suitably cultured human or other mammalian cells. Thispermeability parameter can be expressed as an apparent permeability,P_(app), representing the permeability of the particular cell monolayerto a compound of interest. Any suitably culturable mammalian cellmonolayer can be used to determine its permeability for a particularcompound of interest, although certain cell lines are frequently usedfor this purpose. For instance, the Caco-2 cell line is a human colonadenocarcinoma that can be used as a monolayer culture test system fordetermination of membrane permeability towards compounds of theinvention. In certain embodiments, the synthetic peptide amides of theinvention have a P_(app) of less than about 10⁻⁶ cm/sec. In certainother embodiments, the synthetic peptide amides of the invention have aP_(app) of less than about 10⁻⁷ cm/sec.

In one embodiment, the synthetic peptide amides of the invention at adose of about 3 mg/kg in rat reaches a peak plasma concentration of thesynthetic peptide amide and exhibits at least about a five fold lowerconcentration in brain than such peak plasma concentration.

In another embodiment, the synthetic peptide amides of the inventionhave at least about 50% of maximum efficacy at about 3 hours postadministration of a dose of about 3 mg/kg of the synthetic peptide amidein a rat.

In one embodiment the synthetic peptide amide of the invention exhibitsa long lasting duration of action in a mammal, such as a human. In oneaspect, the synthetic peptide amide has a duration of action that is atleast about 50% of maximum efficacy at 3 hrs post administration of 0.1mg/kg of the synthetic peptide amide. In another aspect the syntheticpeptide amide has a duration of action that is at least about 75% ofmaximum efficacy at 3 hrs post administration of 0.1 mg/kg of thesynthetic peptide amide. In a particular aspect the synthetic peptideamide has a duration of action is at least about 90% of maximum efficacyat 3 hrs post administration of 0.1 mg/kg of the synthetic peptideamide. In a specific aspect, the synthetic peptide amide has a durationof action is at least about 95% of maximum efficacy at 3 hrs postadministration of 0.1 mg/kg of the synthetic peptide amide.

In another embodiment, the invention provides a pharmaceuticalcomposition that includes a synthetic peptide amide according to any ofthe above embodiments and a pharmaceutically acceptable excipient orcarrier. The invention provides methods, compositions, or dosage formsthat employ and/or contain synthetic peptide amides of the inventionthat are selective for the kappa opioid receptor. In particularembodiments, the synthetic peptide amides of the invention exhibit astrong affinity for the kappa opioid receptor and have a high potency askappa opioid receptor agonists.

A pro-drug of a compound such as the synthetic peptide amides of theinvention include pharmaceutically acceptable derivatives which uponadministration can convert through metabolism or other process to abiologically active form of the compound. Pro-drugs are particularlydesirable where the pro-drug has more favorable properties than does theactive compound with respect to bioavailability, stability orsuitability for a particular formulation.

As used herein, a kappa opioid receptor-associated disease, condition ordisorder is any disease, condition or disorder that is preventable ortreatable by activation of a kappa opioid receptor. In one aspect, thesynthetic peptide amides of the invention are kappa opioid receptoragonists that activate the kappa opioid receptor. In some embodiments, aparticular dose and route of administration of the synthetic peptideamide of the invention can be chosen by a clinician to completelyprevent or cure the disease, condition or disorder. In other embodimentsa particular dose and route of administration of the synthetic peptideamide of the invention chosen by the clinician ameliorates or reduce oneor more symptoms of the disease, condition or disorder.

As used herein, “effective amount” or “sufficient amount” of thesynthetic peptide amide of the invention refers to an amount of thecompound as described herein that may be therapeutically effective toinhibit, prevent or treat a symptom of a particular disease, disorder,condition, or side effect. As used herein, a “reduced dose” of a muopioid agonist analgesic compound refers to a dose which when used incombination with a kappa opioid agonist, such as a synthetic peptideamide of the invention, is lower than would be ordinarily provided to aparticular patient, for the purpose of reducing one or more side effectsof the compound. The dose reduction can be chosen such that the decreasein the analgesic or other therapeutic effect of the compound is anacceptable compromise in view of the reduced side effect(s), where saiddecrease in said analgesic or other therapeutic effects of the mu opioidagonist analgesic are at least partially, and most preferably wholly,offset by the analgesic or other therapeutic effect of a syntheticpeptide amide of the invention. Co-administration of a mu opioid agonistanalgesic compound with a synthetic peptide amide of the invention whichacts as a kappa opioid agonist also permits incorporation of a reduceddose of the synthetic peptide amide and/or the mu opioid agonistanalgesic compound to achieve the same therapeutic effect as a higherdose of the synthetic peptide amide or the mu opioid agonist analgesiccompound if administered alone.

As used herein, “pharmaceutically acceptable” refers to compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for contact with the tissues ofhuman beings and animals without severe toxicity, irritation, allergicresponse, or other complications, commensurate with a benefit to-riskratio that is reasonable for the medical condition being treated.

As used herein, “dosage unit” refers to a physically discrete unitsuited as unitary dosages for a particular individual or condition to betreated. Each unit may contain a predetermined quantity of activesynthetic peptide amide compound(s) calculated to produce the desiredtherapeutic effect(s), optionally in association with a pharmaceuticalcarrier. The specification for the dosage unit forms may be dictated by(a) the unique characteristics of the active compound or compounds, andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such active compound orcompounds. The dosage unit is often expressed as weight of compound perunit body weight, for instance, in milligrams of compound per kilogramof body weight of the subject or patient (mg/kg). Alternatively, thedosage can be expressed as the amount of the compound per unit bodyweight per unit time, (mg/kg/day) in a particular dosage regimen. Infurther alternatives, the dosage can be expressed as the amount ofcompound per unit body surface area (mg/m²) or per unit body surfacearea per unit time (mg/m²/day). For topical formulations, the dosage canbe expressed in a manner that is conventional for that formulation,e.g., a one-half inch ribbon of ointment applied to the eye, where theconcentration of compound in the formulation is expressed as apercentage of the formulation.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of compounds wherein the parent compound is modified bymaking acid or base salts thereof. Examples of pharmaceuticallyacceptable salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkali or organic salts ofacidic residues, such as carboxylic acids, and the like. Thepharmaceutically acceptable salts include the conventional non-toxicsalts or the quaternary ammonium salts of the parent compound formed,for example, from non-toxic inorganic or organic acids. For instance,such conventional non-toxic salts include those derived from inorganicacids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric,nitric acids and the like; and the salts prepared from organic acidssuch as acetic, propionic, succinic, glycolic, stearic, lactic, malic,tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionicacids, and the like. These physiologically acceptable salts are preparedby methods known in the art, e.g., by dissolving the free amine baseswith an excess of the acid in aqueous alcohol, or neutralizing a freecarboxylic acid with an alkali metal base such as a hydroxide, or withan amine. Thus, a pharmaceutically acceptable salt of a syntheticpeptide amide can be formed from any such peptide amide having eitheracidic, basic or both functional groups. For example, a peptide amidehaving a carboxylic acid group, may in the presence of apharmaceutically suitable base, form a carboxylate anion paired with acation such as a sodium or potassium cation. Similarly, a peptide amidehaving an amine functional group may, in the presence of apharmaceutically suitable acid such as HCl, form a salt.

One example of a pharmaceutically acceptable solvate of a syntheticpeptide amide is a combination of a peptide amide with solvent moleculeswhich yields a complex of such solvent molecules in association with thepeptide amide. Particularly suitable hydrates of compounds are suchhydrates which either have comparable activity or hydrates which areconverted back to the active compound following administration. Apharmaceutically acceptable N-oxide of a synthetic peptide amide whichcontains an amine is such a compound wherein the nitrogen atom of theamine is bonded to an oxygen atom.

A pharmaceutically acceptable crystalline, isomorphic crystalline oramorphous form of a synthetic peptide amide of the invention can be anycrystalline or non-crystalline form of a pharmaceutically acceptableacidic, basic, zwitterionic, salt, hydrate or any other suitably stable,physiologically compatible form of the synthetic peptide amide accordingto the invention.

The synthetic peptide amides of the invention can be incorporated intopharmaceutical compositions. The compositions can include an effectiveamount of the synthetic peptide amide in a pharmaceutically acceptablediluent, excipient or carrier. Conventional excipients, carriers and/ordiluents for use in pharmaceutical compositions are generally inert andmake up the bulk of the preparation.

In a particular embodiment, the synthetic peptide amide is a kappaopioid receptor agonist. In another embodiment, the synthetic peptideamide is a selective kappa opioid receptor agonist. The target site canbe a kappa receptor in the patient or subject in need of such treatmentor preventative. Certain synthetic peptide amide kappa opioid receptoragonists of the invention are peripherally acting and show little or noCNS effects at therapeutically effective doses.

The pharmaceutical excipient or carrier can be any compatible, non-toxicsubstance suitable as a vehicle for delivery the synthetic peptide amideof the invention. Suitable excipients or carriers include, but are notlimited to, sterile water (preferably pyrogen-free), saline,phosphate-buffered saline (PBS), water/ethanol, water/glycerol,water/sorbitol, water/polyethylene glycol, propylene glycol,cetylstearyl alcohol, carboxymethylcellulose, corn starch, lactose,glucose, microcrystalline cellulose, magnesium stearate,polyvinylpyrrolidone (PVP), citric acid, tartaric acid, oils, fattysubstances, waxes or suitable mixtures of any of the foregoing.

The pharmaceutical composition according to the invention can beformulated as a liquid, semisolid or solid dosage form. For example thepharmaceutical composition can be in the form of a solution forinjection, drops, syrup, spray, suspension, tablet, patch, capsule,dressing, suppository, ointment, cream, lotion, gel, emulsion, aerosolor in a particulate form, such as pellets or granules, optionallypressed into tablets or lozenges, packaged in capsules or suspended in aliquid. The tablets can contain binders, lubricants, diluents, coloringagents, flavoring agents, wetting agents and may be enteric-coated tosurvive the acid environment of the stomach and dissolve in the morealkaline conditions of the intestinal lumen. Alternatively, the tabletscan be sugar-coated or film coated with a water-soluble film.Pharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

Binders include for instance, starch, mucilage, gelatin and sucrose.Lubricants include talc, lycopodium, magnesium and calciumstearate/stearic acid. Diluents include lactose, sucrose, mannitol,salt, starch and kaolin. Wetting agents include propylene glycol andsorbitan monostearate.

As used herein, local application or administration refers toadministration of a pharmaceutical composition according to theinvention to the site, such as an inflamed joint, that exhibits thepainful and/or inflamed condition. Such local application includesintrajoint, such as intra-articular application, via injection,application via catheter or delivery as part of a biocompatible device.Thus, local application refers to application to a discrete internalarea of the body, such as, for example, a joint, soft tissue area (suchas muscle, tendon, ligaments, intraocular or other fleshy internalareas), or other internal area of the body. In particular, as usedherein, local application refers to applications that providesubstantially no systemic delivery and/or systemic administration of theactive agents in the present compositions. Also, as used herein, localapplication is intended to refer to applications to discrete areas ofthe body, that is, other than the various large body cavities (such as,for example, the peritoneal and/or pleural cavities).

As used herein, topical application refers to application to the surfaceof the body, such as to the skin, eyes, mucosa and lips, which can be inor on any part of the body, including but not limited to the epidermis,any other dermis, or any other body tissue. Topical administration orapplication means the direct contact of the pharmaceutical compositionaccording to the invention with tissue, such as skin or membrane,particularly the cornea, or oral, vaginal or anorectal mucosa. Thus, forpurposes herein, topical application refers to application to the tissueof an accessible body surface, such as, for example, the skin (the outerintegument or covering) and the mucosa (the mucus-producing, secretingand/or containing surfaces). In particular, topical application refersto applications that provide little or substantially no systemicdelivery of the active compounds in the present compositions. Exemplarymucosal surfaces include the mucosal surfaces of the eyes, mouth (suchas the lips, tongue, gums, cheeks, sublingual and roof of the mouth),larynx, esophagus, bronchus, trachea, nasal passages, vagina andrectum/anus.

For oral administration, an active ingredient can be administered insolid dosage forms, such as capsules, tablets, and powders, or in liquiddosage forms, such as elixirs, syrups, and suspensions. Activecomponent(s) can be encapsulated in gelatin capsules together withinactive ingredients and powdered carriers, such as glucose, lactose,sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesiumstearate, stearic acid, sodium saccharin, talcum, magnesium carbonateand the like. Examples of additional inactive ingredients that may beadded to provide desirable color, taste, stability, buffering capacity,dispersion or other known desirable features are red iron oxide, silicagel, sodium lauryl sulfate, titanium dioxide, edible white ink and thelike. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, or entericcoated for selective disintegration in the gastrointestinal tract.Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. To facilitate drug stabilityand absorption, peptides of the invention can be released from a capsuleafter passing through the harsh proteolytic environment of the stomach.Methods for enhancing peptide stability and absorption after oraladministration are well known in the art (e.g., Mahato R I. Emergingtrends in oral delivery of peptide and protein drugs. Critical Reviewsin Therapeutic Drug Carrier Systems. 20:153-214, 2003).

Dosage forms such as lozenges, chewable tablets and chewing gum permitmore rapid therapeutic action compared to per-oral dosage forms of thesynthetic peptide amide compounds of the invention having significantbuccal absorption. Chewing gum formulations are solid, single dosepreparations with a base consisting mainly of gum, that are intended tobe chewed but not swallowed, and contain one or more compounds of theinvention which are released by chewing and are intended to be used forlocal treatment of pain and inflammation of the mouth or systemicdelivery after absorption through the buccal mucosa. See for example,U.S. Pat. No. 6,322,828 to Athanikar and Gubler entitled: Process formanufacturing a pharmaceutical chewing gum.

For nasal administration, the peripherally selective kappa opioidreceptor agonists can be formulated as aerosols. The term “aerosol”includes any gas-borne suspended phase of the compounds of the instantinvention which is capable of being inhaled into the bronchioles ornasal passages. Specifically, aerosol includes a gas-borne suspension ofdroplets of the compounds of the instant invention, as may be producedin a metered dose inhaler or nebulizer, or in a mist sprayer. Aerosolalso includes a dry powder composition of a compound of the instantinvention suspended in air or other carrier gas, which may be deliveredby insufflation from an inhaler device, for example. See Ganderton &Jones, Drug Delivery to the Respiratory Tract, Ellis Horwood (1987);Gonda (1990) Critical Reviews in Therapeutic Drug Carrier Systems6:273-313; and Raeburn et al. (1992) J. Pharmacol. Toxicol. Methods27:143-159.

The pharmaceutical compositions of the invention can be prepared in aformulation suitable for systemic delivery, such as for instance byintravenous, subcutaneous, intramuscular, intraperitoneal, intranasal,transdermal, intravaginal, intrarectal, intrapulmonary or oral delivery.Alternatively, the pharmaceutical compositions of the invention can besuitably formulated for local delivery, such as, for instance, fortopical, or iontophoretic delivery, or for transdermal delivery by apatch coated, diffused or impregnated with the formulation, and localapplication to the joints, such as by intra-articular injection.

Preparations for parenteral administration include sterile solutionsready for injection, sterile dry soluble products ready to be combinedwith a solvent just prior to use, including hypodermic tablets, sterilesuspensions ready for injection, sterile dry insoluble products ready tobe combined with a vehicle just prior to use and sterile emulsions. Thesolutions may be either aqueous or nonaqueous, and thereby formulatedfor delivery by injection, infusion, or using implantable pumps. Forintravenous, subcutaneous, and intramuscular administration, usefulformulations of the invention include microcapsule preparations withcontrolled release properties (R. Pwar et al. Protein and peptideparenteral controlled delivery. Expert Opin Biol Ther. 4(8):1203-12,2004) or encapsulation in liposomes, with an exemplary form beingpolyethylene coated liposomes, which are known in the art to have anextended circulation time in the vasculature (e.g. Koppal, T. “Drugdelivery technologies are right on target”, Drug Discov. Dev. 6, 49-50,2003).

For ophthalmic administration, the present invention provides a methodof treating glaucoma or ophthalmic pain and inflammation, comprisingadministering to an eye of a patient in need thereof a therapeuticallyeffective amount of a synthetic peptide amide of the invention. Thesynthetic peptide amide can be administered topically with aneye-compatible pharmaceutical carrier or non-systemically using acontact lens or intraocular implant that can optionally contain polymersthat provide sustained release of the synthetic peptide amide. Sucheye-compatible pharmaceutical carriers can include adjuvants,antimicrobial preservatives, surfactants, and viscolyzers etc. It isknown in the art that high concentrations of many compounds are irritantto the eye and low concentrations are less irritant; thus theformulation is often designed to include the lowest effectiveconcentrations of active compound, preservative, surfactant, and/orviscolyzer, said viscolyzer preferably having a high surface tension toreduce irritation of the eye while increasing the retention ofophthalmic solutions at the eye surface. Such controlled release of thesynthetic peptide amides of the invention can last 6 months to a yearfor implants, or for shorter periods (3-14 days) for contact lenses.Such implants can be osmotic pumps, biodegradable matrices, orintraocular sustained release devices. Such topical compositions caninclude a buffered saline solution with or without liposomes.

Aqueous polymeric solutions, aqueous suspensions, ointments, and gelscan be used for topical formulations of the synthetic peptide amides ofthe invention for ocular applications. The aqueous formulations may alsocontain liposomes for creating a reservoir of the synthetic peptideamide. Certain of these topical formulations are gels which enhancepre-corneal retention without the inconvenience and impairment of visionassociated with ointments. The eye-compatible pharmaceutical carrier canalso include a biodegradable synthetic polymer. Biodegradablemicrosphere compositions approved for human use include thepolylactides: poly(lactic acid), poly(glycolic acid), andpoly(lactic-coglycolic) acid. Additional biodegradable formulationsinclude, but are not limited to: poly(anhydride-co-imide),poly(lactic-glycolic acid), polyethyl-2-cyanoacrylate, polycaprolactone,polyhydroxybutyrate valerate, polyorthoester, andpolyethylene-oxide/polybutylene teraphthalate. Intraocular implantationor injection of sustained release compositions that include a syntheticpeptide amide of the invention can provide long-term control (rangingfrom months to years) of intraocular pressure, and thereby avoiding orreducing the need for topical preparations. Useful methods forformulating and dispensing ophthalmic medications are disclosed in U.S.Pat. No. 7,122,579 to Schwartz et al, and in U.S. Pat. No. 7,105,512 toMorizono et al. Methods for formulating ophthalmic medications incontact lenses are disclosed by Gulsen and Chauhan, Ophthalmic drugdelivery through contact lenses. Investigative Ophthalmology and VisualScience, (2004) 45:2342-2347.

Preparations for transdermal delivery are incorporated into a devicesuitable for said delivery, said device utilizing, e.g., iontophoresis(Kalia Y N et al. Iontophoretic Drug Delivery. Adv Drug Deliv Rev.56:619-58, 2004) or a dermis penetrating surface (Prausnitz M R.Microneedles for Transdermal Drug Delivery. Adv Drug Deliv Rev.56:581-7, 2004), such as are known in the art to be useful for improvingthe transdermal delivery of drugs. An electrotransport device andmethods of operation thereof are disclosed in U.S. Pat. No. 6,718,201.Methods for the use of iontophoresis to promote transdermal delivery ofpeptides are disclosed in U.S. Pat. No. 6,313,092 and U.S. Pat. No.6,743,432.

As used herein the terms “electrotransport”, “iontophoresis”, and“iontophoretic” refer to the delivery through a body surface (e.g., skinor mucosa) of one or more pharmaceutically active compounds by means ofan applied electromotive force to an agent containing reservoir. Thecompound may be delivered by electromigration, electroporation,electroosmosis or any combination thereof. Electroosmosis has also beenreferred to as electrohydrokinesis, electro convection, and electricallyinduced osmosis. In general, electroosmosis of a compound into a tissueresults from the migration of solvent in which the compound iscontained, as a result of the application of electromotive force to thetherapeutic species reservoir, such as for instance, solvent flowinduced by electromigration of other ionic species. During theelectrotransport process, certain modifications or alterations of theskin may occur such as the formation of transiently existing pores inthe skin, also referred to as “electroporation.” Any electricallyassisted transport of species enhanced by modifications or alterationsto the body surface (e.g., formation of pores in the skin) are alsoincluded in the term “electrotransport” as used herein. Thus, as usedherein, applied to the compounds of the instant invention, the terms“electrotransport”, “iontophoresis” and “iontophoretic” refer to (1) thedelivery of charged agents by electromigration, (2) the delivery ofuncharged agents by the process of electroosmosis, (3) the delivery ofcharged or uncharged agents by electroporation, (4) the delivery ofcharged agents by the combined processes of electromigration andelectroosmosis, and/or (5) the delivery of a mixture of charged anduncharged agents by the combined processes of electromigration andelectroosmosis. Electrotransport devices generally employ twoelectrodes, both of which are positioned in close electrical contactwith some portion of the skin of the body. One electrode, called theactive or donor electrode, is the electrode from which the therapeuticagent is delivered into the body. The other electrode, called thecounter or return electrode, serves to close the electrical circuitthrough the body. In conjunction with the patient's skin, the circuit iscompleted by connection of the electrodes to a source of electricalenergy, e.g., a battery, and usually to circuitry capable of controllingcurrent passing through the device.

Depending upon the electrical charge of the compound to be deliveredtransdermally, either the anode or cathode may be the active or donorelectrode. Thus, if the compound to be transported is positivelycharged, e.g., the compound exemplified in Example 1 herein, then thepositive electrode (the anode) will be the active electrode and thenegative electrode (the cathode) will serve as the counter electrode,completing the circuit. However, if the compound to be delivered isnegatively charged, then the cathodic electrode will be the activeelectrode and the anodic electrode will be the counter electrode.Electrotransport devices additionally require a reservoir or source ofthe therapeutic agent that is to be delivered into the body. Such drugreservoirs are connected to the anode or the cathode of theelectrotransport device to provide a fixed or renewable source of one ormore desired species or agents. Each electrode assembly is comprised ofan electrically conductive electrode in ion-transmitting relation withan ionically conductive liquid reservoir which in use is placed incontact with the patient's skin. Gel reservoirs such as those describedin Webster (U.S. Pat. No. 4,383,529) are one form of reservoir sincehydrated gels are easier to handle and manufacture than liquid-filledcontainers. Water is one liquid solvent that can be used in suchreservoirs, in part because the salts of the peptide compounds of theinvention are water soluble and in part because water is non-irritatingto the skin, thereby enabling prolonged contact between the hydrogelreservoir and the skin. For electrotransport, the synthetic peptides ofthe invention can be formulated with flux enhancers such as ionicsurfactants or cosolvents other than water (See for example, U.S. Pat.No. 4,722,726 and European Patent Application 278,473, respectively).Alternatively the outer layer (i.e., the stratum corneum) of the skincan be mechanically disrupted prior to electrotransport deliverytherethrough, for example as described in U.S. Pat. No. 5,250,023.

Peripherally synthetic peptide amides that are well suited forelectrotransport can be selected by measuring their electrotransportflux through the body surface (e.g., the skin or mucosa), e.g., ascompared to a standardized test peptide with known electrotransport fluxcharacteristics, e.g. thyrotropin releasing hormone (R. Burnette et al.J. Pharm. Sci. (1986) 75:738) or vasopressin (Nair et al. Pharmacol Res.48:175-82, 2003). Transdermal electrotransport flux can be determinedusing a number of in vivo or in vitro methods well known in the art. Invitro methods include clamping a piece of skin of an appropriate mammal(e.g., human cadaver skin) between the donor and receptor compartmentsof an electrotransport flux cell, with the stratum corneum side of theskin piece facing the donor compartment. A liquid solution or gelcontaining the drug to be delivered is placed in contact with thestratum corneum, and electric current is applied to electrodes, oneelectrode in each compartment. The transdermal flux is calculated bysampling the amount of drug in the receptor compartment. Two successfulmodels used to optimize transdermal electrotransport drug delivery arethe isolated pig skin flap model (Heit M C et al. Transdermaliontophoretic peptide delivery: in vitro and in vivo studies withluteinizing hormone releasing hormone. J. Pharm. Sci. 82:240-243, 1993),and the use of isolated hairless skin from hairless rodents or guineapigs, for example. See Hadzija B W et al. Effect of freezing oniontophoretic transport through hairless rat skin. J. Pharm. Pharmacol.44, 387-390, 1992. Compounds of the invention for transdermaliontophoretic delivery can have one, or typically, two chargednitrogens, to facilitate their delivery.

Other useful transdermal delivery devices employ high velocity deliveryunder pressure to achieve skin penetration without the use of a needle.Transdermal delivery can be improved, as is known in the art, by the useof chemical enhancers, sometimes referred to in the art as “permeationenhancers”, i.e., compounds that are administered along with the drug(or in some cases used to pretreat the skin, prior to drugadministration) in order to increase the permeability of the stratumcorneum, and thereby provide for enhanced penetration of the drugthrough the skin. Chemical penetration enhancers are compounds that areinnocuous and serve merely to facilitate diffusion of the drug throughthe stratum corneum, whether by passive diffusion or an energy drivenprocess such as electrotransport. See, for example, Meidan V M et al.Enhanced iontophoretic delivery of buspirone hydrochloride across humanskin using chemical enhancers. Int. J. Pharm. 264:73-83, 2003.

Pharmaceutical dosage forms for rectal administration include rectalsuppositories, capsules and tablets for systemic effect. Rectalsuppositories as used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesinclude bases or vehicles and agents that raise the melting point of thesuppositories. Examples of bases include cocoa butter (theobroma oil),glycerin-gelatin, carbowax, (polyoxyethylene glycol) and appropriatemixtures of mono-, di- and triglycerides of fatty acids. Combinations ofthe various bases can also be used. Agents that raise the melting pointof suppositories include spermaceti and wax. Rectal suppositories may beprepared either by the compression method or by molding. Rectalsuppositories typically weigh about 2 gm to about 3 gm. Tablets andcapsules for rectal administration are manufactured using the samepharmaceutically acceptable substance(s) and by the same methods as forformulations for oral administration.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include sodium chloride for injection,Ringers solution for injection, isotonic dextrose for injection, sterilewater for injection, dextrose and lactated Ringers solution forinjection. Nonaqueous parenteral vehicles include fixed oils ofvegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.Antimicrobial agents in bacteriostatic or fungistatic concentrationsmust be added to parenteral preparations packaged in multiple dosecontainers which include phenols or cresols, mercurials, benzyl alcohol,chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters,thimerosal, benzalkonium chloride and benzethonium chloride. Isotonicagents include sodium chloride and dextrose. Buffers include phosphateand citrate. Antioxidants include sodium bisulfite. Local anestheticsinclude procaine hydrochloride. Suspending and dispersing agents includesodium carboxymethylcelluose, hydroxypropyl methylcellulose andpolyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (Tween80). A sequestering or chelating agent of metal ions such as EDTA canalso be incorporated. Pharmaceutical carriers also include ethylalcohol, polyethylene glycol and propylene glycol for water misciblevehicles and the pH can be adjusted to a physiologically compatible pHby addition of sodium hydroxide, hydrochloric acid, citric acid orlactic acid.

The active ingredient may be administered all at once, or may be dividedinto a number of smaller doses to be administered at intervals of time,or as a controlled release formulation. The term “controlled releaseformulation” encompasses formulations that allow the continuous deliveryof a synthetic peptide amide of the invention to a subject over a periodof time, for example, several days to weeks. Such formulations may beadministered subcutaneously or intramuscularly and allow for thecontinual steady state release of a predetermined amount of compound inthe subject over time. The controlled release formulation of syntheticpeptide amide may be, for example, a formulation of drug containingpolymeric microcapsules, such as those described in U.S. Pat. Nos.4,677,191 and 4,728,721, incorporated herein by reference. Theconcentration of the pharmaceutically active compound is adjusted sothat administration provides an effective amount to produce a desiredeffect. The exact dose depends on the age, weight and condition of thepatient or animal, as is known in the art. For any particular subject,specific dosage regimens can be adjusted over time according to theindividual need and the professional judgment of the personadministering or supervising the administration of the formulations.Thus, the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedinvention.

The unit dose parenteral preparations include packaging in an ampoule orprepackaged in a syringe with, or without a needle for delivery. Allpreparations for parenteral administration are typically sterile, as ispracticed in the art. Illustratively, intravenous infusion of a sterileaqueous buffered solution containing an active compound is an effectivemode of administration. In another embodiment a sterile aqueous or oilysolution or suspension containing the active material can be injected asnecessary to produce the desired pharmacological effect.

The pharmaceutical compositions of the invention can be delivered oradministered intravenously, transdermally, transmucosally, intranasally,subcutaneously, intramuscularly, orally or topically (such as forexample to the eye). The compositions can be administered forprophylactic treatment of individuals suffering from, or at risk of adisease or a disorder. For therapeutic applications, a pharmaceuticalcomposition is typically administered to a subject suffering from adisease or disorder, in an amount sufficient to inhibit, prevent, orameliorate the disease or disorder. An amount adequate to accomplishthis is defined as a “therapeutically effective dose.”

The pharmaceutical compositions of the invention can be administered toa mammal for prophylactic or therapeutic purposes in any of theabove-described formulations and delivery modes. The mammal can be anymammal, such as a domesticated or feral mammal, or even a wild mammal.The mammal can be any primate, ungulate, canine or feline. For instance,and without limitation, the mammal may be a pet or companion animal,such as a dog or a cat; a high-value mammal such as a thoroughbred horseor a show animal; a farm animal, such as a cow, a goat, a sheep or pig;or a primate such as an ape, gorilla, orangutan, lemur, monkey orchimpanzee. A suitable mammal for prophylaxis or treatment using thepharmaceutical compositions of the invention is a human.

The pharmaceutical compositions of the invention can be administered toa mammal having a disease or condition treatable by activation of thekappa opioid receptor. Alternatively, the pharmaceutical compositionscan be administered as prophylactics to a mammal having a risk ofcontracting or developing a disease or condition preventable byactivation of the kappa opioid receptor. Diseases or conditions that canbe treated or prevented by administration of the pharmaceuticalcompositions of the invention include, without limitation, any conditionthat can be ameliorated by activation of the kappa opioid receptor,including such conditions as pain, inflammation, pruritis, hyponatremia,hypokalemia, congestive heart failure, liver cirrhosis, nephroticsyndrome, hypertension, edema, ileus, tussis and glaucoma.

In a particular embodiment, the pharmaceutical compositions of theinvention can be co-administered with or can include one or more othertherapeutic compounds or adjuvants, such as but not limited to otheropioids, cannabinoids, antidepressants, anticonvulsants, neuroleptics,antihistamines, acetaminophen, corticosteroids, ion channel blockingagents, non-steroidal anti-inflammatory drugs (NSAIDs), and diuretics,many of which are synergistic in effect with the synthetic peptideamides of the invention.

Suitable opioids, include, without limitation, alfentanil, alphaprodine,anileridine, bremazocine, buprenorphine, butorphanol, codeine,conorphone, dextromoramide, dextropropoxyphene, dezocine, diamorphine,dihydrocodeine, dihydromorphine, diphenoxylate, dipipanone, doxpicomine,ethoheptazine, ethylketazocine, ethylmorphine, etorphine, fentanyl,hydrocodone, hydromorphone, ketobemidone, levomethadyl, levorphanol,lofentanil, loperamide, meperidine (pethidine), meptazinol, methadone,morphine, morphine-6-glucuronide, nalbuphine, nalorphine, nicomorphine,oxycodone, oxymorphone, pentazocine, phenazocine, phenoperidine,piritramide, propiram, propoxyphene, remifentanil, sufentanil, tilidate,tonazocine, and tramadol.

One embodiment of the invention is co-formulation and/orco-administration of an opioid with substantial agonist activity at themu opioid receptor, such as morphine, fentanyl, hydromorphone, oroxycodone, together with a synthetic peptide amide of the invention, forthe purpose of a mu opioid dose-sparing effect, where the dose of the muopioid is reduced to minimize common mu opioid side effects,particularly in opioid-naïve patients. Such side effects includeconstipation, nausea, vomiting, sedation, respiratory depression,pruritis (itching), mental confusion, disorientation and cognitiveimpairment, urinary retention, biliary spasm, delirium, myoclonic jerks,and seizures. The selection of the reduced mu opioid dose requiresexpert clinical judgment, and depends on the unique characteristics ofthe various mu opioids, as well as patient characteristics such as painintensity, patient age, coexisting disease, current drug regimen andpotential drug interactions, prior treatment outcomes, and patientpreference (McCaffery, M. and Pasero, C., Pain Clinical Manual, SecondEdition, Mosby, 1999).

Cannabinoids suitable for administration with or incorporation into thepharmaceutical compositions of the invention, include any naturalcannabinoid, such as for instance, tetrahydrocannabinol (THC), or a THCderivative, or a synthetic cannabinoid, such as, for instance,levonantradol, marinol, nabilone, rimonabant or savitex.

Suitable antidepressants that can be co-administered with orincorporated into the pharmaceutical compositions of the invention,include for example, tricyclic antidepressants such as imipramine,desipramine, trimipramine, protriptyline, nortriptyline, amitriptyline,doxepin, and clomipramine; atypical antidepressants such as amoxapine,maprotiline, trazodone, bupropion, and venlafaxine; serotonin-specificreuptake inhibitors such as fluoxetine, sertraline, paroxetine,citalopram and fluvoxamine; norepinephrine-specific reuptake inhibitorssuch as reboxetine; or dual-action antidepressants such as nefazodoneand mirtazapine.

Suitable neuroleptics that can be co-administered with or incorporatedinto the pharmaceutical compositions of the invention, include anyneuroleptic, for example, a compound with D2 dopamine receptorantagonist activity such as domperidone, metaclopramide, levosulpiride,sulpiride, thiethylperazine, ziprasidone, zotepine, clozapine,chlorpromazine, acetophenazine, carphenazine, chlorprothixene,fluphenazine, loxapine, mesoridazine, molindone, perphenazine, pimozide,piperacetazine, perchlorperazine, thioridazine, thiothixene,trifluoperazine, triflupromazine, pipamperone, amperozide, quietiapine,melperone, remoxipride, haloperidol, rispiridone, olanzepine,sertindole, ziprasidone, amisulpride, prochlorperazine, and thiothixene.

Anticonvulsants such as phenobarbital, phenytoin, primidone,carbamazepine, ethosuximide, lamotrigine, valproic acid, vigabatrin,felbamate, gabapentin, levetiracetam, oxcarbazepine, remacemide,tiagabine, and topiramate can also usefully be incorporated into thepharmaceutical compositions of the invention.

Muscle relaxants such as methocarbamol, orphenadrine, carisoprodol,meprobamate, chlorphenesin carbamate, diazepam, chlordiazepoxide andchlorzoxazone; anti-migraine agents such as sumitriptan, analeptics suchas caffeine, methylphenidate, amphetamine and modafinil; antihistaminessuch as chlorpheniramine, cyproheptadine, promethazine and pyrilamine,as well as corticosteroids such as methylprednisolone, betamethasone,hydrocortisone, prednisolone, cortisone, dexamethasone, prednisone,alclometasone, clobetasol, clocortrolone, desonide, desoximetasone,diflorasone, fluocinolone, fluocinonide, flurandrenolide, fluticasone,floromethalone, halcinonide, halobetasol, loteprednol, mometasone,prednicarbate, and triamcinolone can also be incorporated into thepharmaceutical compositions of the invention.

Ion channel blocking agents such as, for instance, the sodium ionchannel blocker, carbamazepine, as commonly used in the treatment oftinnitus, arrhythmia, ischemic stroke and epilepsy can beco-administered with or incorporated into the pharmaceuticalcompositions of the invention. Alternatively, or in addition, calciumion channel blockers, such as ziconotide, can also be used, as canantagonists of the ion channel associated with the NMDA receptor, suchas ketamine. There is evidence that at least some of these ion channelblockers can potentiate the analgesic effects of the kappa agonist andthereby reduce the dose required for affective pain relief. See forinstance, Wang et al., 2000, Pain 84: 271-81.

Suitable NSAIDs, or other non-opioid compounds with anti-inflammatoryand/or analgesic activity, that can be co-administered with orincorporated into the pharmaceutical compositions of the inventioninclude, but are not limited to one or more of the following:aminoarylcarboxylic acid derivatives such as etofenamate, meclofenamicacid, mefanamic acid, niflumic acid; arylacetic acid derivatives such asacemetacin, amfenac, cinmetacin, clopirac, diclofenac, fenclofenac,fenclorac, fenclozic acid, fentiazac, glucametacin, isoxepac, lonazolac,metiazinic acid, naproxin, oxametacine, proglumetacin, sulindac,tiaramide and tolmetin; arylbutyric acid derivatives such as butibufenand fenbufen; arylcarboxylic acids such as clidanac, ketorolac andtinoridine. arylpropionic acid derivatives such as bucloxic acid,carprofen, fenoprofen, flunoxaprofen, ibuprofen, ibuproxam, oxaprozin,phenylalkanoic acid derivatives such as flurbiprofen, piketoprofen,pirprofen, pranoprofen, protizinic acid and tiaprofenic acid;pyranocarboxylic acids such as etodolac; pyrazoles such as mepirizole;pyrazolones such as clofezone, feprazone, mofebutazone, oxyphinbutazone,phenylbutazone, phenyl pyrazolidininones, suxibuzone andthiazolinobutazone; salicylic acid derivatives such as aspirin,bromosaligenin, diflusinal, fendosal, glycol salicylate, mesalamine,1-naphthyl salicylate, magnesium salicylate, olsalazine andsalicylamide, salsalate, and sulfasalazine; thiazinecarboxamides such asdroxicam, isoxicam and piroxicam others such as ε-acetamidocaproic acid,acetaminophen, s-adenosylmethionine, 3-amino-4-hydroxybutyric acid,amixetrine, bendazac, bucolome, carbazones, cromolyn, difenpiramide,ditazol, hydroxychloroquine, indomethacin, ketoprofen and its activemetabolite 6-methoxy-2-naphthylacetic acid; guaiazulene, heterocylicaminoalkyl esters of mycophenolic acid and derivatives, nabumetone,nimesulide, orgotein, oxaceprol, oxazole derivatives, paranyline,pifoxime, 2-substituted-4,6-di-tertiary-butyl-s-hydroxy-1,3-pyrimidines, proquazone and tenidap,and cox-2 (cyclooxygenase II) inhibitors, such as celecoxib orrofecoxib.

Suitable diuretics that can be co-administered with or incorporated intothe pharmaceutical preparations of the invention, include, for example,inhibitors of carbonic anhydrase, such as acetazolamide,dichlorphenamide, and methazolamide; osmotic diuretics, such asglycerin, isosorbide, mannitol, and urea; inhibitors of Na⁺—K⁺-2Cl⁻symport (loop diuretics or high-ceiling diuretics), such as furosemide,bumetanide, ethacrynic acid, torsemide, axosemide, piretanide, andtripamide; inhibitors of Na⁺—Cl⁻ symport (thiazide and thiazidelikediuretics), such as bendroflumethiazide, chlorothiazide,hydrochlorothiazide, hydroflumethazide, methyclothiazide, polythiazide,trichlormethiazide, chlorthalidone, indapamide, metolazone, andquinethazone; and, in addition, inhibitors of renal epithelial Na⁺channels, such as amiloride and triamterene, antagonists ofmineralocorticoid receptors (aldosterone antagonists), such asspironolactone, canrenone, potassium canrenoate, and eplerenone, which,together, are also classified as K⁺-sparing diuretics. One embodiment isco-formulation and/or co-administration of a loop or thiazide diuretictogether with a synthetic peptide amide of the invention for the purposeof a loop or thiazide diuretic dose-sparing effect, wherein the dose ofthe loop or thiazide diuretic is reduced to minimize undesired waterretention, and prevent or reduce hyponatremia, particularly in thecontext of congestive heart failure, as well as other medical conditionswhere decreasing body fluid retention and normalizing sodium balancecould be beneficial to a patient in need thereof. See R M Reynolds etal. Disorders of sodium balance Brit. Med. J. 2006; 332:702-705.

The kappa opioid receptor-associated hyponatremia can be any disease orcondition where hyponatremia (low sodium condition) is present, e.g., inhumans, when the sodium concentration in the plasma falls below 135mmol/L, an abnormality that can occur in isolation or, more frequently,as a complication of other medical conditions, or as a consequence ofusing medications that can cause sodium depletion.

A further embodiment is co-formulation and/or co-administration of apotassium-sparing diuretic, e.g., a mineralocorticoid receptorantagonist, such as spironolactone or eplerenone, together with asynthetic peptide amide of the invention, for the purpose of enabling areduced dose of said potassium-sparing diuretic, wherein the dose ofsaid diuretic is reduced to minimize hyperkalemia or metabolic acidosis,e.g., in patients with hepatic cirrhosis.

In particular embodiments, the synthetic peptide amides of the inventionexhibit a long lasting duration of action when administered intherapeutically relevant doses in vivo. For instance, in someembodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 3 mg/kg of the synthetic peptideamide maintain at least about 50% of maximum efficacy in a kappa opioidreceptor-dependent assay at 3 hours post administration. In certainother embodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg of the synthetic peptideamide maintain at least about 50% of maximum efficacy in a kappa opioidreceptor-dependent assay at 3 hours post administration. The maximumefficacy is operationally defined as the highest level of efficacydetermined for the particular kappa opioid receptor-dependent assay forall agonists tested.

In certain embodiments, the synthetic peptide amides of the inventionwhen administered to a mammal at a dose of 0.1 mg/kg maintain at leastabout 75% of maximum efficacy at 3 hours post administration. In stillother embodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg maintain at least about90% of maximum efficacy at 3 hours post administration. In certain otherembodiments, the synthetic peptide amides of the invention whenadministered to a mammal at a dose of 0.1 mg/kg maintain at least about95% of maximum efficacy at three hours post administration.

The invention further provides a method of treating or preventing akappa opioid receptor-associated disease or condition in a mammal,wherein the method includes administering to the mammal a compositioncontaining an effective amount of a synthetic peptide amide of theinvention. The mammal can be any mammal, such as a domesticated or feralmammal, or even a wild mammal. Alternatively, the mammal can be aprimate, an ungulate, a canine or a feline. For instance, and withoutlimitation, the mammal may be a pet or companion animal, such as ahigh-value mammal such as a thoroughbred or show animal; a farm animal,such as a cow, a goat, a sheep or pig; or a primate such as an ape ormonkey. In one particular aspect, the mammal is a human.

The effective amount can be determined according to routine methods byone of ordinary skill in the art. For instance, an effective amount canbe determined as a dosage unit sufficient to prevent or to treat a kappareceptor-associated disease or condition in the mammal. Alternatively,the effective amount may be determined as an amount sufficient toapproximate the EC₅₀ concentration or an amount sufficient toapproximate two or three times or up to about five or even about tentimes the EC₅₀ concentration in a therapeutically relevant body fluid ofthe mammal, for instance, where the body fluid is in direct appositionto a target tissue, such as the synovial fluid of an inflamed joint in apatient suffering from rheumatoid arthritis.

In one embodiment the synthetic peptide amide of the invention is apharmaceutical composition that includes an effective amount of thesynthetic peptide amide of the invention and a pharmaceuticallyacceptable excipient or carrier. In one aspect, the pharmaceuticalcomposition includes a synthetic peptide amide of the invention in anamount effective to treat or prevent a kappa opioid receptor-associatedcondition in a mammal, such as a human. In another aspect the kappaopioid receptor-associated condition is pain, inflammation, pruritis,edema, ileus, tussis or glaucoma.

In one embodiment the pharmaceutical composition of the inventionfurther includes one or more of the following compounds: an opioid, acannabinoid, an antidepressant, an anticonvulsant, a neuroleptic, acorticosteroid, an ion channel blocking agent or a non-steroidalanti-inflammatory drug (NSAID).

Pharmaceutical compositions that include a synthetic peptide amide ofthe invention and a pharmaceutically acceptable vehicle or carrier canbe used to treat or prevent one or more of a variety of kappa opioidreceptor-associated diseases, disorders or conditions.

The kappa opioid receptor-associated disease, disorders or conditionpreventable or treatable with the synthetic peptide amides of theinvention can be any kappa opioid receptor-associated condition,including but not limited to acute or chronic pain, inflammation,pruritis, hyponatremia, edema, ileus, tussis and glaucoma. For instance,the kappa opioid receptor-associated pain can be neuropathic pain,somatic pain, visceral pain or cutaneous pain. Some diseases, disorders,or conditions are associated with more than one form of pain, e.g.,postoperative pain can have any or all of neuropathic, somatic,visceral, and cutaneous pain components, depending upon the type andextent of surgical procedure employed.

The kappa opioid receptor-associated inflammation can be anyinflammatory disease or condition including, but not limited tosinusitis, rheumatoid arthritis tenosynovitis, bursitis, tendonitis,lateral epicondylitis, adhesive capsulitis, osteomyelitis,osteoarthritic inflammation, inflammatory bowel disease (IBD), irritablebowel syndrome (IBS), ocular inflammation, otitic inflammation orautoimmune inflammation.

The kappa opioid receptor-associated pruritis can be any pruriticdisease or condition such as, for instance, ocular pruritis, e.g.,associated with conjunctivitis, otitic pruritis, pruritis associatedwith end-stage renal disease, where many patients are receiving kidneydialysis, and other forms of cholestasis, including primary biliarycirrhosis, intrahepatic cholestasis of pregnancy, chronic cholestaticliver disease, uremia, malignant cholestasis, jaundice, as well asdermatological conditions such as eczema (dermatitis), including atopicor contact dermatitis, psoriasis, polycythemia vera, lichen planus,lichen simplex chronicus, pediculosis (lice), thyrotoxicosis, tineapedis, urticaria, scabies, vaginitis, anal pruritis associated withhemorrhoids and, as well as insect bite pruritis and drug-inducedpruritis, such as mu opioid-induced pruritis.

The kappa opioid receptor-associated edema can be any edematous diseaseor condition such as, for instance, edema due to congestive heartdisease or to a syndrome of inappropriate antidiuretic hormone (ADH)secretion.

The kappa opioid receptor-associated ileus can be any ileus disease orcondition including, but not limited post-operative ileus oropioid-induced bowel dysfunction.

The kappa opioid receptor-associated neuropathic pain can be anyneuropathic pain, such as, for instance, trigeminal neuralgia, diabeticpain, viral pain such as herpes zoster-associated pain,chemotherapy-induced pain, nerve-encroaching metastatic cancer pain,neuropathic pain associated with traumatic injury and surgicalprocedures, as well as variants of headache pain that are thought tohave a neuropathic component, e.g., migraine.

Kappa opioid-associated pain also includes ocular pain, e.g., followingphoto-refractive keratectomy (PRK), ocular laceration, orbital floorfracture, chemical burns, corneal abrasion or irritation, or associatedwith conjunctivitis, corneal ulcers, scleritis, episcleritis,sclerokeratitis, herpes zoster ophthalmicus, interstitial keratitis,acute iritis, keratoconjunctivitis sicca, orbital cellulites, orbitalpseudotumor, pemphigus, trachoma, or uveitis.

Kappa opioid-associated pain also includes throat pain, particularlyassociated with inflammatory conditions, such as allergic rhinitis,acute bronchitis, the common cold, contact ulcers, herpes simplex virallesions, infectious mononucleosis, influenza, laryngeal cancer, acutelaryngitis, acute necrotizing ulcerative gingivitis, peritonsillarabscess, pharyngeal burns, pharyngitis, reflus laryngopharyngitis, acutesinusitis, and tonsillitis.

In addition, kappa opioid receptor-associated pain can be arthriticpain, kidney-stone, urinary tract stone, gallstone, and bile duct stonepain, uterine cramping, dysmenorrhea, endometriosis, mastitis,dyspepsia, post-surgical pain (such as, for instance, from appendectomy,open colorectal surgery, hernia repair, prostatectomy, colonicresection, gastrectomy, splenectomy, colectomy, colostomy, pelviclaparoscopy, tubal ligation, hysterectomy, vasectomy or cholecystecomy),post medical procedure pain (such as, for instance, after colonoscopy,cystoscopy, hysteroscopy or cervical or endometrial biopsy), otiticpain, breakthrough cancer pain, and pain associated with a GI disordersuch as IBD or IBS or other inflammatory conditions, particularly of theviscera (e.g., gastroesophageal reflux disease, pancreatitis, acutepolynephritis, ulcerative colitis, acute pyelonephritis, cholecystitis,cirrhosis, hepatic abscess, hepatitis, duodenal or gastric ulcer,esophagitis, gastritis, gastroenteritis, colitis, diverticulitis,intestinal obstruction, ovarian cyst, pelvic inflammatory disease,perforated ulcer, peritonitis, prostatitis, interstitial cystitis), orexposure to toxic agents, such as insect toxins, or drugs such assalicylates or NSAIDs.

The present invention provides a method of treating or preventing akappa opioid receptor-associated disease or condition in a mammal, suchas a human, wherein the method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention. In another embodiment the kappa opioidreceptor-associated condition is pain, inflammation (such as rheumatoidarthritic inflammation, osteoarthritic inflammation, IBD inflammation,IBS inflammation, ocular inflammation, otitic inflammation or autoimmuneinflammation), pruritis (such as atopic dermatitis,kidney-dialysis-associated pruritis, ocular pruritis, otitic pruritis,insect bite pruritis, or opioid-induced pruritis), edema, ileus, tussisor glaucoma. In one aspect, the pain is a neuropathic pain (such astrigeminal neuralgia, migraine, diabetic pain, viral pain,chemotherapy-induced pain or metastatic cancer pain), a somatic pain, avisceral pain or a cutaneous pain. In another aspect the pain isarthritic pain, kidney-stone pain, uterine cramping, dysmenorrhea,endometriosis, dyspepsia, post-surgical pain, post medical procedurepain, ocular pain, otitic pain, breakthrough cancer pain or painassociated with a GI disorder, such as IBD or IBS. In another aspect thepain is pain associated with surgery, wherein the surgery is pelviclaparoscopy, tubal ligation, hysterectomy and cholecystecomy.Alternatively, the pain can be pain associated with a medical procedure,such as for instance, colonoscopy, cystoscopy, hysteroscopy orendometrial biopsy. In a specific aspect, the atopic dermatitis can bepsoriasis, eczema or contact dermatitis. In another specific aspect, theileus is post-operative ileus or opioid-induced bowel dysfunction.

Kappa opioid receptor-associated pain includes hyperalgesia, which isbelieved to be caused by changes in the milieu of the peripheral sensoryterminal occur secondary to local tissue damage. Tissue damage (e.g.,abrasions, burns) and inflammation can produce significant increases inthe excitability of polymodal nociceptors (C fibers) and high thresholdmechanoreceptors (Handwerker et al. (1991) Proceeding of the VIth WorldCongress on Pain, Bond et al., eds., Elsevier Science Publishers BV, pp.59-70; Schaible et al. (1993) Pain 55:5-54). This increased excitabilityand exaggerated responses of sensory afferents is believed to underliehyperalgesia, where the pain response is the result of an exaggeratedresponse to a stimulus. The importance of the hyperalgesic state in thepost-injury pain state has been repeatedly demonstrated and appears toaccount for a major proportion of the post-injury/inflammatory painstate. See for example, Woold et al. (1993) Anesthesia and Analgesia77:362-79; Dubner et al. (1994) In, Textbook of Pain, Melzack et al.,eds., Churchill-Livingstone, London, pp. 225-242.

In another embodiment the kappa opioid receptor-associated condition ispain, inflammation (such as rheumatoid arthritic inflammation,osteoarthritic inflammation, IBD inflammation, IBS inflammation, ocularinflammation, otitic inflammation or autoimmune inflammation), pruritis(such as atopic dermatitis, kidney-dialysis-associated pruritis, ocularpruritis, otitic pruritis, insect bite pruritis, or opioid-inducedpruritis), edema, ileus, tussis or glaucoma. In one aspect, the pain isa neuropathic pain (such as trigeminal neuralgia, migraine, diabeticpain, viral pain, chemotherapy-induced pain or metastatic cancer pain),a somatic pain, a visceral pain or a cutaneous pain. In another aspectthe pain is arthritic pain, kidney-stone pain, uterine cramping,dysmenorrhea, endometriosis, dyspepsia, post-surgical pain, post medicalprocedure pain, ocular pain, otitic pain, breakthrough cancer pain orpain associated with a GI disorder, such as IBD or IBS. In anotheraspect the pain is pain associated with surgery, wherein the surgery ispelvic laparoscopy, tubal ligation, hysterectomy and cholecystecomy.Alternatively, the pain can be pain associated with a medical procedure,such as for instance, colonoscopy, cystoscopy, hysteroscopy orendometrial biopsy. In a specific aspect, the atopic dermatitis can bepsoriasis, eczema or contact dermatitis. In another specific aspect, theileus is post-operative ileus or opioid-induced bowel dysfunction.

In another embodiment the kappa opioid receptor-associated condition isa kappa opioid receptor-associated condition preventable or treatable bysodium and potassium-sparing diuresis, also known as aquaresis. Anexample of such kappa opioid receptor-associated conditions preventableor treatable by administering a synthetic peptide amide of the inventionincludes edema. The edema may be due to any of a variety of diseases orconditions, such as congestive heart disease or syndrome ofinappropriate ADH secretion.

In another embodiment the kappa opioid receptor-associated condition ishyponatremia or other edematous disease. The kappa opioidreceptor-associated hyponatremia or edema can be any hyponatremic oredematous disease or condition such as, for instance, hyponatremia andedema associated with congestive heart failure or to a syndrome ofinappropriate antidiuretic hormone (ADH) secretion, or hyponatremia thatis associated with intensive diuretic therapy with thiazides and/or loopdiuretics. The synthetic peptide amides of the invention exhibit asignificant sodium-sparing and potassium-sparing aquaretic effect, whichis beneficial in the treatment of edema-forming pathological conditionsassociated with hyponatremia and/or hypokalemia. Accordingly, thesynthetic peptide amides of the invention also have utility in methodsof treating or preventing hyponatremia-related conditions, examples ofwhich are provided below. Hyponatremia-related conditions can becategorized according to volume status as hypervolemic, euvolemic, orhypovolemic.

Hypervolemic hyponatremia is usually caused by an increase in total bodywater level as may be observed in cases of congestive heart failure,nephrotic syndrome and hepatic cirrhosis.

Euvolemic hyponatremia is often found in the syndrome of inappropriateantidiuretic hormone (ADH) secretion and may also be associated withpneumonia, small-cell lung cancer, polydipsia, cases of head injury, andorganic causes (e.g., use of certain drugs, such as haloperidol) or apsychogenic cause.

Hypovolemic hyponatremia is due to a relative decrease in total bodysodium level and may be associated with diuretic use, cases ofinterstitial nephritis or excessive sweating.

These forms of hyponatremia can be further classified according to theconcentration of sodium in the urine (i.e., whether the concentration isgreater than or less than thirty millimoles per liter. See: R M Reynoldset al. Disorders of sodium balance, Brit. Med. J. 2006; 332:702-705.

The kappa opioid receptor-associated hyponatremia can be any disease orcondition where hyponatremia (low sodium condition) is present, e.g., inhumans, when the sodium concentration in the plasma falls below 135mmol/L, an abnormality that can occur in isolation or, more frequently,as a complication of other medical conditions, or as a consequence ofusing medications that can cause sodium depletion.

In addition to these conditions, numerous other conditions areassociated with hyponatremia including, without limitation: neoplasticcauses of excess ADH secretion, including carcinomas of lung, duodenum,pancreas, ovary, bladder, and ureter, thymoma, mesothelioma, bronchialadenoma, carcinoid, gangliocytoma and Ewing's sarcoma; infections suchas: pneumonia (bacterial or viral), abscesses (lung or brain),cavitation (aspergillosis), tuberculosis (lung or brain), meningitis(bacterial or viral), encephalitis and AIDS; vascular causes such as:cerebrovascular occlusions or hemorrhage and cavernous sinus thrombosis;neurologic causes such as: Guillan-Barre syndrome, multiple sclerosis,delirium tremens, amyotrophic lateral sclerosis, hydrocephalus,psychosis, peripheral neuropathy, head trauma (closed and penetrating),CNS tumors or infections and CNS insults affecting hypothalamicosmoreceptors; congenital malformations including: agenesis of corpuscallosum, cleftlip/palate and other midline defects; metabolic causessuch as: acute intermittent porphyria, asthma, pneurothorax andpositive-pressure respiration; drugs such as: thiazide diuretics,acetaminophen, barbiturates, cholinergic agents, estrogen, oralhypoglycemic agents, vasopressin or desmopressin, high-dose oxytocin,chlorpropamide, vincristine, carbamezepine, nicotine, phenothiazines,cyclophosphamide, tricyclic antidepressants, monoamine oxidaseinhibitors and serotonin reuptake inhibitors; administration of excesshypotonic fluids, e.g., during hospitalization, surgery, or during orafter athletic events (i.e., exercise-associated hyponatremia), as wellas use of low-sodium nutritional supplements in elderly individuals. Seefor example, Harrison's Principles of Internal Medicine, 16th Ed.(2005), p. 2102.

Other conditions associated with hyponatremia include renal failure,nephrotic syndrome (membranous nephropathy and minimal change disease),cachexia, malnutrition, rhabdomyolysis, surgical procedures, electivecardiac catheterization, blood loss, as well as hypercalcemia,hypokalemia, and hyperglycemia with consequent glycosuria leading toosmotic diuresis.

The invention also provides a method of treating or preventing aneurodegenerative disease or condition in a mammal, such as a human,wherein the method includes administering to the mammal a compositionthat includes an effective amount of a synthetic peptide amide asdescribed above. The neurodegenerative disease or condition can be anyneurodegenerative disease or condition, such as for instance, ischemia,anoxia, stroke, brain injury, spinal cord injury or reperfusion injury.Alternatively, the neurodegenerative disease or condition can be aneurodegenerative disease of the eye. Particular neurodegenerativediseases of the eye treatable or preventable by the method of theinvention include glaucoma, macular degeneration, retinal ischemicdisease and diabetic neuropathy.

In certain embodiments the invention provides methods of prevention ortreatment of certain neuronal diseases and conditions, such as diseasesand conditions having a neurodegenerative component. Synthetic peptideamides of the invention can be administered in an amount effective toprotect neuronal cells against the effects of pathology or injury thatwould lead to neurodegeneration and/or neuronal cell death of theuntreated cells. For example, several diseases or conditions of the eyethat have a neurodegenerative component can be prevented or treated byadministration of an effective amount of the synthetic peptide amides ofthe invention. Such diseases and conditions of the eye include glaucoma,macular degeneration, retinal ischemic disease and diabetic neuropathy.Progression of these diseases and conditions is believed to involveneurodegeneration or neuronal cell death, for example by programmed celldeath (apoptosis) in which the neuronal cells are committed to a pathwaythat without intervention would lead to cell death. It has been foundthat development or progression of these diseases and conditions can beprevented, or at least slowed, by treatment with kappa opioid receptoragonists. This improved outcome is believed to be due to neuroprotectionby the kappa opioid receptor agonists. See for instance, Kaushik et al.“Neuroprotection in Glaucoma” (2003) J. Postgraduate Medicine vol. 49(1): pp. 90-95.

In the case of glaucoma it is believed that prophylaxis and treatment byadministration of kappa opioid receptor agonists is mediated by at leasttwo distinct activities induced by activation of the kappa opioidreceptor: neuroprotection and reduction of intraocular pressure (IOP).While not wishing to be bound by theory, it is believed thatneuroprotection is due, at least in part, to induction of atrialnatriuretic peptide (ANP) in the eye, leading to protection againstoxidative damage and other insults.

Abnormally high intraocular pressure is also believed to be a factorleading to the development of glaucoma. Elevated intraocular pressurecan also be prevented or treated by administration of kappa opioidreceptor agonists by three separate activities triggered by activationof the receptor: reduction in secretion of aqueous humor, increasedoutflow of aqueous humor and aquaresis (sodium- and potassium-sparingdiuresis, resulting in loss of water).

The invention also provides a method of treating or preventing akappa-receptor-associated disease or condition of the eye of a mammal,such as high intraocular pressure (IOP). The method includesadministering to the mammal a composition that includes an effectiveamount of a synthetic peptide amide as described above. In one aspect ofthe invention, the synthetic peptide amide is administered topically. Inanother aspect, the synthetic peptide amide is administered as animplant.

In other embodiments the invention provides methods of prevention ortreatment of certain cardiovascular diseases and conditions having acellular degenerative component. Synthetic peptide amides of theinvention can be administered in an amount effective to protectmyocardial cells against the effects of pathology or injury that wouldlead to degeneration and/or cell death of the untreated cells. Forexample, several cardiovascular diseases or conditions can be preventedor treated by administration of an effective amount of the syntheticpeptide amides of the invention. Such cardiovascular diseases andconditions include, without limitation, coronary heart disease,ischemia, cardiac infarct, reperfusion injury and arrhythmia. See forexample, Wu et al. “Cardioprotection of Preconditioning by MetabolicInhibition in the Rat Ventricular Myocyte—Involvement of kappa OpioidReceptor” (1999) Circulation Res vol. 84: pp. 1388-1395. See also Yu etal. “Anti-Arrhythmic Effect of kappa Opioid Receptor Stimulation in thePerfused Rat Heart: Involvement of a cAMP-Dependent Pathway” (1999) JMol Cell Cardiol. vol. 31(10): pp. 1809-1819.

Diseases and conditions of other tissues and organs that can beprevented or treated by administration of an effective amount of thesynthetic peptide amides of the invention include, but are not limitedto ischemia, anoxia, stroke, brain or spinal cord injury and reperfusioninjury.

Another form of kappa opioid receptor-associated pain treatable orpreventable with the synthetic peptide amides of the invention ishyperalgesia. In one embodiment, the method includes administering aneffective amount of a synthetic peptide amide of the invention to amammal suffering from or at risk of developing hyperalgesia to prevent,ameliorate or completely alleviate the hyperalgesia.

The synthetic peptide amides of the invention can be administered bymethods disclosed herein for the treatment or prevention of anyhyperalgesic condition, such as, but without limitation, a hyperalgesiccondition associated with allergic dermatitis, contact dermatitis, skinulcers, inflammation, rashes, fungal irritation and hyperalgesicconditions associated with infectious agents, burns, abrasions, bruises,contusions, frostbite, rashes, acne, insect bites/stings, skin ulcers,mucositis, gingivitis, bronchitis, laryngitis, sore throat, shingles,fungal irritation, fever blisters, boils, Plantar's warts, surgicalprocedures or vaginal lesions. For instance, the synthetic peptideamides of the invention can be administered topically to a mucosalsurface, such as the mouth, esophagus or larynx, or to the bronchial ornasal passages. Alternatively, the synthetic peptide amides of theinvention can be administered topically to the vagina or rectum/anus.

Moreover, the synthetic peptide amides of the invention can beadministered by methods disclosed herein for the treatment or preventionof any hyperalgesic condition associated with burns, abrasions, bruises,abrasions (such as corneal abrasions), contusions, frostbite, rashes,acne, insect bites/stings, skin ulcers (for instance, diabetic ulcers ora decubitus ulcers), mucositis, inflammation, gingivitis, bronchitis,laryngitis, sore throat, shingles, fungal irritation (such as athlete'sfoot or jock itch), fever blisters, boils, Plantar's warts or vaginallesions (such as vaginal lesions associated with mycosis or sexuallytransmitted diseases). Methods contemplated for administration of thesynthetic peptide amides of the invention for the treatment orprevention of hyperalgesia include those wherein the compound istopically applied to a surface in the eyes, mouth, larynx, esophagus,bronchial, nasal passages, vagina or rectum/anus.

Hyperalgesic conditions associated with post-surgery recovery can alsobe addressed by administration of the synthetic peptide amides of theinvention. The hyperalgesic conditions associated with post-surgeryrecovery can be any hyperalgesic conditions associated with post-surgeryrecovery, such as for instance, radial keratectomy, tooth extraction,lumpectomy, episiotomy, laparoscopy and arthroscopy.

Hyperalgesic conditions associated with inflammation are alsoaddressable by administration of the synthetic peptide amides of theinvention. Periodontal inflammation, orthodontic inflammation,inflammatory conjunctivitis, hemorrhoids and venereal inflammations canbe treated or prevented by topical or local administration of thesynthetic peptide amides of the invention.

The invention also provides a method of inducing diuresis in a mammal inneed thereof. The method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention as described above.

The invention further provides a method of inducing prolactin secretionin a mammal. The method includes administering to the mammal acomposition comprising an effective amount of a synthetic peptide amideof the invention as described above. The method of inducing prolactinsecretion is suitable for treating a mammal, such as a human sufferingfrom insufficient lactation, inadequate lactation, sub-optimallactation, reduced sperm motility, an age-related disorder, type Idiabetes, insomnia or inadequate REM sleep. In a particular aspect, themethod includes co-administering the synthetic peptide amide with areduced dose of a mu opioid agonist analgesic compound to produce atherapeutic analgesic effect, the compound having an associated sideeffect, wherein the reduced dose of the compound has a lower associatedside effect than the side effect associated with the dose of the muopioid agonist analgesic compound necessary to achieve the therapeuticanalgesic effect when administered alone.

The present invention also provides a method of binding a kappa opioidreceptor in a mammal, the method includes the step of administering tothe mammal a composition containing an effective amount of a syntheticpeptide amide of the present invention. The effective amount can bedetermined according to routine methods by one of ordinary skill in theart. For instance, the effective amount can be determined as a dosageunit sufficient to bind kappa opioid receptors in a mammal and cause anantinociceptive effect, an anti-inflammatory effect, an aquareticeffect, or an elevation of serum prolactin levels or any other kappaopioid receptor-responsive effect. Alternatively, the effective amountmay be determined as an amount sufficient to approximate the EC₅₀ in abody fluid of the mammal, or an amount sufficient to approximate two orthree, or up to about five or even about ten times the EC₅₀ in atherapeutically relevant body fluid of the mammal.

Synthesis of the Peptide Amides of the Invention

As used herein, the chemical designation“tetrapeptide-[ω(4-amino-piperidine-4-carboxylic acid)]” is used toindicate the aminoacyl moiety of the synthetic peptide amides of theinvention derived from 4-aminopiperidine-4-carboxylic acid, wherein thenitrogen atom of the piperidine ring is bound to the C-terminalcarbonyl-carbon of the tetrapeptide fragment, unless otherwiseindicated.

FIG. 1 shows the general synthetic scheme used in the preparation ofcompounds (1) (6), (7), (10) and (11); FIG. 2 shows the generalsynthetic scheme used in the preparation of compounds (2) to (5), (8),(9) and (12)-(14); FIG. 3 shows the general synthetic scheme used in thepreparation of synthetic peptide amides (15)-(24); and FIG. 4 shows thescheme used in the preparation of the synthetic peptide amides(25)-(37):

Compound (1): D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[4-Amidinohomopiperazineamide] (SEQ ID NO: 1):

Compound (2): D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 2):

Compound (3):D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 1):

Compound (4): D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]-OH(SEQ ID NO: 2):

Compound (5): D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]-OH(SEQ ID NO: 3):

Compound (6):D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]-OH (SEQ IDNO: 1):

Compound (7): D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide] (SEQ ID NO:4):

Compound (8): D-Phe-D-Phe-D-Leu-D-Har-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 3):

Compound (9):D-Phe-D-Phe-D-Leu-(ε-iPr)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 5):

Compound (10):D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 6):

Compound (11): D-Phe-D-Phe-D-Leu-D-Nar-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 7):

Compound (12): D-Phe-D-Phe-D-Leu-D-Dbu-[N-(4-piperidinyl)-L-proline]-OH(SEQ ID NO: 8):

Compound (13): D-Phe-D-Phe-D-Leu-D-Nar-[N-(4-piperidinyl)-L-proline]-OH(SEQ ID NO: 7):

Compound (14):D-Phe-D-Phe-D-Leu-D-Dap(amidino)-[N-(4-piperidinyl)-L-proline]-OH (SEQID NO: 6):

Compound (15): D-Phe-D-Phe-D-Leu-D-Lys-[4-Amidinohomopiperazine amide](SEQ ID NO: 2):

Compound (16): D-Phe-D-Phe-D-Leu-D-Har-[4-Amidinohomopiperazine amide](SEQ ID NO: 3):

Compound (17): D-Phe-D-Phe-D-Leu-(ε-iPr)D-Lys-[4-Amidinohomopiperazineamide] (SEQ ID NO: 5):

Compound (18):D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[4-Amidinohomopiperazine amide] (SEQID NO: 6):

Compound (19):D-Phe-D-Phe-D-Nle-(β-amidino)D-Dap-[4-Amidinohomopiperazine amide] (SEQID NO: 6):

Compound (20): D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[homopiperazine amide](SEQ ID NO: 6):

Compound (21): D-Phe-D-Phe-D-Nle-(β-amidino)D-Dap-[homopiperazine amide](SEQ ID NO: 6):

Compound (22): D-Phe-D-Phe-D-Leu-D-Dbu-[4-Amidinohomopiperazine amide](SEQ ID NO: 8):

Compound (23): D-Phe-D-Phe-D-Leu-D-Nar-[4-Amidinohomopiperazine amide](SEQ ID NO: 7):

Compound (24): D-Phe-D-Phe-D-Leu-D-Arg-[4-Amidinohomopiperazine amide](SEQ ID NO: 4):

Compound (25): D-Phe-D-Phe-D-Leu-D-Lys-[2,8-diazaspiro[4,5]decan-1-oneamide] (SEQ ID NO: 2):

Compound (26):D-Phe-D-Phe-D-Leu-D-Lys-[2-methyl-2,8-diazaspiro[4,5]decan-1-one amide](SEQ ID NO: 2):

Compound (27):D-Phe-D-Phe-D-Leu-D-Lys-[1,3,8-triazaspiro[4,5]decane-2,4-dione amide](SEQ ID NO: 2):

Compound (28):D-Phe-D-Phe-D-Leu-D-Lys-[5-chloro-1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3)H-oneamide] (SEQ ID NO: 2):

Compound (29):D-Phe-D-Phe-D-Leu-D-Lys-[morpholino(piperidin-4-yl)methanone amide] (SEQID NO: 2):

Compound (30):D-Phe-D-Phe-D-Leu-D-Lys-[4-phenyl-1-(piperidin-yl-1H-imidazol-2(3H)-oneamide] (SEQ ID NO: 2):

Compound (31):D-Phe-D-Phe-D-Leu-D-Lys-[4-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)piperidineamide] (SEQ ID NO: 2):

Compound (32): D-Phe-D-Phe-D-Leu-D-Lys-[1-(piperidin-4-yl)indolin-2-oneamide] (SEQ ID NO: 2):

Compound (33):D-Phe-D-Phe-D-Leu-D-Lys-[1-phenyl-1,3,8-triazaspiro[4.5]decan-4-oneamide] (SEQ ID NO: 2):

Compound (34):D-Phe-D-Phe-D-Leu-D-Lys-[imidazo[1,2-a]pyridine-2-ylmethyl amide] (SEQID NO: 2):

Compound (35) D-Phe-D-Phe-D-Leu-D-Lys-[(5-methylpyrazin-2-yl)methylamide] (SEQ ID NO: 2):

Compound (36):D-Phe-D-Phe-D-Leu-D-Lys-[1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-oneamide] (SEQ ID NO: 2):

Compound (37):D-Phe-D-Phe-D-Leu-D-Lys-[4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridineamide] (SEQ ID NO: 2):

EXAMPLES General Experimental Synthetic Methods

Amino acid derivatives and resins were purchased from commercialproviders (Novabiochem, Bachem, Peptide International and PepTechCorporation). Other chemicals and solvents were purchased fromSigma-Aldrich, Fisher Scientific and VWR. The compounds herein weresynthesized by standard methods in solid phase peptide chemistryutilizing both Fmoc and Boc methodology. Unless otherwise specified, allreactions were performed at room temperature.

The following standard references provide guidance on generalexperimental setup, and the availability of required starting materialand reagents: Kates, S. A., Albericio, F., Eds., Solid Phase Synthesis,A Practical Guide, Marcel Dekker, New York, Basel, (2000); Bodanszky,M., Bodanszky, A., Eds., The Practice of Peptide Synthesis, SecondEdition, Springer-Verlag, (1994); Atherton, E., Sheppard, R. C., Eds.,Solid Phase Peptide Synthesis, A Practical Approach, IRL Press at OxfordUniversity Press, (1989); Stewart, J. M., Young, J. D., Solid PhaseSynthesis, Pierce Chemical Company, (1984); Bisello, et al., J. Biol.Chem. 273, 22498-22505 (1998); and Merrifield, R. B., J. Am. Chem. Soc.85, 2149-2154 (1963).

Additional abbreviations used herein:

-   -   ACN: acetonitrile    -   Aloc: allyloxycarbonyl    -   Boc: tert-butoxycarbonyl    -   BOP: benzotriazole-1-yl-oxy-tris(dimethylamino)-phosphonium        hexafluorophosphate    -   Cbz: benzyloxycarbonyl.    -   Cbz-OSu: Na-(Benzyloxycarbonyloxy) succinimide    -   DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene    -   DCM: Dichloromethane    -   Dde: 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl    -   DIC: N,N′-diisopropylcarbodiimide    -   DIEA: N,N-diisopropylethylamine    -   DMF: N,N-dimethylformamide    -   Fmoc: 9-fluorenylmethoxycarbonyl    -   HATU: 2-(1H-9-azabenzotriazole-1-yl)-1,1,3,3-tetramethylaminium        hexafluorophosphate    -   HBTU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium        hexafluorophosphate    -   HOBt: 1-hydroxybenzotriazole    -   HPLC: high performance liquid chromatography    -   i: iso    -   ivDde:        1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl    -   NMM: 4-methyl morpholino    -   NMP: N-methylpyrrolidinone    -   All: allyl    -   o-NBS—Cl: o-nitrobenzenesulfonyl chloride    -   Pbf: 2,2,4,6,7-pentamethyldihydro-benzofuran-5-sulfonyl    -   PyBOP: benzotriazole-1-yloxy-tris-pyrrolidino-phosphonium        hexafluorophosphate    -   RP: reversed phase    -   TB TU: 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium        tetrafluoroborate    -   TEAP: triethylammonium phosphate    -   TFA: trifluoroacetic acid    -   TIS: triisopropylsilane    -   TMOF: trimethyl orthoformate    -   TMSOTf: trimethylsilyl trifluoromethanesulfonate    -   Trt: trityl

Peptides synthesized by Fmoc methodology were cleaved with a mixture ofTFA/TIS/H₂O (v/v/v=95:2.5:2.5). The cleavage step in the Boc methodologywas accomplished either with a mixture of HF/anisole (v/v=9:1) or with amixture of TMSOTf/TFA/m-cresol (v/v/v=2:7:1).

Coupling reactions in peptide chain elongation were carried out eithermanually on a peptide synthesizer and mediated by coupling reagents witha 2 to 4-fold excess amino acid derivatives. The coupling reagents usedin the synthesis of the various compounds of the invention were chosenfrom the following combinations: DIC/HOBt, HATU/DIEA, HBTU/DIEA,TBTU/DIEA, PyBOP/DIEA, and BOP/DIEA.

Deprotection of the side chain of amino acid in position No. 4(designated Xaa₄ in the final synthetic peptide amide product) of resinbound peptides was achieved as follows: Peptides were assembled startingfrom Xaa₄ and progressively adding Xaa₃, then Xaa₂ and finally, Xaa₁.The side chain protecting groups of the diamino acid introduced at Xaa₄were selectively removed as follows: (i) N-Dde or N-ivDde groups wereremoved by 2-4% hydrazine in DMF. See Chabra, S. R., et al., TetrahedronLett. 39:1603-1606 (1998) and Rohwedder, B., et al., Tetrahedron Lett.,39: 1175 (1998); (ii) N-Aloc: removed by 3 eq. (Ph₃P)₄Pd inCHCl₃/AcOH/NMM (v/v/v=37:2:1). See Kates, S. A., et al. in “PeptidesChemistry, Structure and Biology, Proc. 13^(th) American PeptideSymposium”, Hodges, R. S. and Smith, J. A. (Eds), ESCOM, Leiden, 113-115(1994).

When peptides were assembled with Boc protection methodology, the sidechain protecting group of the diamino acids introduced at Xaa₄ wasN-Fmoc, which was removed by 20-30% piperidine in DMF.

Isopropylation of the terminal nitrogen on the side chain of amino acidat Xaa₄ of resin bound peptides was achieved as follows: Afterdeprotection, the resin bound peptide with the free ω-amino function atXaa₄ was reacted with a mixture of acetone and NaBH(OAc)₃ in TMOFproducing the resin bound Nω-isopropyl peptide.

Monomethylation of the terminal nitrogen on the side chain of amino acidat Xaa₄ of resin bound peptides: To synthesize resin bound Nω-methylpeptides, the free ω-amino function was first derivatized witho-nitrobenzene-sulfonyl chloride (o-NBS—Cl; B iron, E.; Chatterjee, J.;Kessler, H. Optimized selective N-methylation of peptides on solidsupport. J. Pep. Sci. 12:213-219 (2006). The resulting sulfonamide wasthen methylated with a mixture of dimethylsulphate and1,8-diaza-bicyclo[5.4.0]undec-7-ene in NMP. The o-NBS protecting groupwas subsequently removed by a mixture of mercaptoethanol and1,8-diazabicyclo[5.4.0]undec-7-ene in NMP.

Guanylation of the terminal nitrogen on the side chain of amino acid atXaa₄ of resin bound peptides: After deprotection, the resin boundpeptide with the free ω-amino function in position No. 4 was reactedwith a mixture of 1H-pyrazole-1-carboxamidine hydrochloride(Bernatowicz, M. S., et al., J. Org. Chem. 57, 2497-2502 (1992) and DIEAin DMF producing the resin bound Nω-guanidino peptide.

Peptides were purified by preparative HPLC in triethylammonium phosphate(TEAP) or trifluoroacetic acid (TFA) buffers. When required, thecompounds were finally converted to trifluoroacetate or acetate saltsusing conventional HPLC methodology. Fractions with purity exceeding 97%were pooled and lyophilized. Purity of the synthesized peptides wasdetermined by analytical RP-HPLC.

Analytical RP-HPLC was performed on a Waters 600 multisolvent deliverysystem with a Waters 486 tunable absorbance UV detector and a Waters 746data module. HPLC analyses of peptides were carried out using a VydacC₁₈ column (0.46×25 cm, 5 μm particle size, 300 Å pore size) at a flowrate of 2.0 ml/min. Solvents A and B were 0.1% TFA in H₂O and 0.1% TFAin 80% ACN/20% H₂O, respectively. Retention times (t_(R)) are given inmin. Preparative RP-HPLC was accomplished using a Vydac C₁₈ preparativecartridge (5×30 cm, 15-20 μm particle size, 300 Å pore size) at a flowrate of 100 ml/min, on a Waters Prep LC 2000 preparative chromatographsystem with a Waters 486 tunable absorbance UV detector and a Servogor120 strip chart recorder. Buffers A and B were 0.1% TFA in H₂O and 0.1%TFA in 60% ACN/40% H₂O, respectively. HPLC analysis of the finalcompound was performed on a Hewlett Packard 1090 Liquid Chromatographusing a Phenomenex Synergi MAX-RP C₁₂ column (2.0×150 mm, 4 μM particlesize, 80 Å pore size) at a flow rate of 0.3 ml/min at 40° C. Buffers Aand B were 0.01% TFA in H₂O and 0.01% TFA in 70% ACN/30% H₂O,respectively. The identity of the synthetic peptide amides was confirmedby electrospray mass spectrometry. Mass spectra were recorded on aFinnigan LCQ mass spectrometer with an ESI source.

Example 1 Synthesis of Compound (1)

D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[4-Amidinohomopiperazine amide] (SEQ IDNO: 1).

See the scheme shown in FIG. 1. The amino acid derivatives used wereBcz-D-Phe-OH, Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, and Fmoc-D-Lys(Dde)-OH. Thefully protected resin bound peptide was synthesized manually startingfrom p-nitrophenylcarbonate Wang resin (5.0 g, 4.4 mmol; Novabiochem).The attachment of homopiperazine to the resin was achieved by mixing itwith a solution of homopiperazine (8.7 g, 87 mmol; Acros Organics) inDCM (100 mL) overnight at room temperature. The resin was washed withDMF and DCM and dried in vacuo. The resulting homopiperazine carbamateWang resin (5.1 g; homopiperazine-[carbamate Wang resin]) was split intoseveral portions and a portion of 1.5 g (1.3 mmol) was used to continuethe peptide synthesis. DIC/HOBt mediated single couplings were performedwith a 3-fold excess of amino acid derivatives. The Fmoc group wasremoved with 25% piperidine in DMF. Upon completion of peptide chainelongation, the resin was treated with 4% hydrazine in DMF for 3×3 minfor Dde removal. The resin was washed with DMF and DCM and dried invacuo. The resulting peptide resin (2.4 g;Bcz-D-Phe-D-Phe-DLeu-DLys-homopiperazine-[carbamate Wang resin]) wassplit again and a portion of 0.6 g (0.3 mmol) was used for subsequentderivatization (N-methylation).

Methylation of the ω-amino function of D-Lys at Xaa₄ was carried out inthree steps: (i) [o-NBS Protection]: The resin-bound peptide (0.3 mmol)was first treated with a solution o-NBS—Cl (0.4 g, 2 mmol) and collidine(0.7 ml, 5 mmol) in NMP (7 ml) at room temperature for 30 min. The resinwas then washed with NMP. (ii) [N-Methylation]: The resin-bound o-NBSprotected peptide was then reacted with a solution of1,8-diazabicyclo[5.4.0]undec-7-ene (0.5 ml, 3 mmol) and dimethylsulfate(1.0 ml, 10 mmol; Aldrich) in NMP (7 ml) at room temperature for 5 min.The resin was then washed with NMP and the washing process was repeatedonce. (iii) [o-NBS Deprotection]: The peptide resin was treated with asolution of mercaptoethanol (0.7 ml, 10 mmol) and1,8-diazabicyclo[5.4.0]undec-7-ene (0.8 ml, 5 mmol) in NMP (7 ml) atroom temperature for 5 min. The resin was then washed with NMP and thewashing process was repeated once.

To protect the resulting N-methyl secondary amine of D-Lys at Xaa₄, theresin-bound methylated peptide was reacted with a solution of Bcz-OSu (6mmol) in DMF (7 ml). The resin was washed with DMF and DCM and dried invacuo. The peptide was then cleaved from the resin by treatment with asolution of TFA/DCM (15 ml, v/v=1:1) at room temperature for 2 hours.The resin was then filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (0.3 mmol;Bcz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Bcz)-[homopiperazine amide]) wasprecipitated from diethyl ether.

For guanylation of the homopiperazine at the C-terminus, the abovepeptide (0.3 mmol) was treated with a solution of1H-Pyrazole-1-carboxamidine hydrochloride (0.4 g, 3.0 mmol) and DIEA(0.5 ml, 6 mmol) in DMF (3 ml) overnight at room temperature. Aceticacid and H₂O were added to quench the reaction and the solution wasfrozen and dried on a lyophilizer to give the desired protected peptide,Bcz-D-Phe-D-Phe-D-Leu-D-Lys(Me,Z)-[4-Amidinohomopiperazine amide] (0.6g).

For final deprotection/hydrolysis, the above peptide (0.6 g) was treatedwith a mixture of TMSOTf/TFA/m-cresol (10 ml, v/v/v=2:7:1) at roomtemperature for 2 hours. The mixture was evaporated and the crudepeptide (0.6 g) was precipitated from diethyl ether.

For purification, the above-derived crude peptide (0.6 g) was dissolvedin 0.1% TFA in H₂O (50 ml) and the solution was loaded onto an HPLCcolumn and purified using TFA buffer system (buffers A=0.1% TFA in H₂Oand B=0.1% TFA in 60% ACN/40% H₂O). The compound was eluted with alinear gradient of buffer B, 25% B to 75% B over 30 min, t_(R)=37% B.The fractions with purity exceeding 97% were pooled, frozen, and driedon a lyophilizer to give the purified peptide as white amorphous powder(153 mg). HPLC analysis: t_(R)=14.41 min, purity 99.8%, gradient 5% B to25% B over 20 min; MS (M+H⁺): expected molecular ion mass 692.5,observed 692.5.

Example 2 Synthesis of Compound (2)

D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylic acid)]-OH (SEQID NO: 2):

See the scheme of FIG. 2 and Biron et al., Optimized selectiveN-methylation of peptides on solid support. J. Peptide Science 12:213-219 (2006). The amino acid derivatives used were Boc-D-Phe-OH,Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Dde)-OH, andN-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid. HPLC and MS analyseswere performed as described in the synthesis of compound (1) describedabove.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (1.8 g, 0.9 mmol; PeptideInternational). Attachment of N-Boc-amino-(4-N-Fmoc-piperidinyl)carboxylic acid followed by peptide chain elongation and deprotection ofDde in D-Lys(Dde) at Xaa₄ was carried out according to the proceduredescribed in the synthesis of compound (1). See above. The resultingpeptide resin (0.9 mmol;Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin]) was split and a portion of 0.3 mmol was used forsubsequent cleavage. The peptide resin (0.3 mmol) was then treated witha mixture of TFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperaturefor 90 min. The resin was then filtered and washed with TFA. Thefiltrate was evaporated in vacuo and the crude peptide (0.3 mmol;D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylic acid)]-OH) wasprecipitated from diethyl ether.

For purification, the crude peptide (0.3 mmol) was dissolved in 2%acetic acid in H₂O (50 ml) and the solution was loaded onto an HPLCcolumn and purified using TEAP buffer system with a pH 5.2 (buffersA=TEAP 5.2 and B=20% TEAP 5.2 in 80% ACN). The compound was eluted witha linear gradient of buffer B, 7% B to 37% B over 60 min. Fractions withpurity exceeding 95% were pooled and the resulting solution was dilutedwith two volumes of water. The diluted solution was then loaded onto anHPLC column for salt exchange and further purification with a TFA buffersystem (buffers A=0.1% TFA in H₂O and B=0.1% TFA in 80% ACN/20% H₂O) anda linear gradient of buffer B, 2% B to 75% B over 25 min. Fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer toyield the purified peptide as white amorphous powder (93 mg). HPLCanalysis: t_(R)=16.43 min, purity 99.2%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 680.4, observed 680.3.

Compound (2) was also prepared using a reaction scheme analogous to thatshown in FIG. 2 with the following amino acid derivatives:Fmoc-D-Phe-OH, Fmoc-D-Leu-OH, Fmoc-D-Lys(Boc)-OH, andBoc-4-amino-1-Fmoc-(piperidine)-4-carboxylic acid.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (PS 1% DVB, 500 g, 1 meq/g).The resin was treated withBoc-4-amino-1-Fmoc-4-(piperidine)-4-carboxylic acid (280 g, 600 mmol) ina mixture of DMF, DCM and DMA (260 mL of each) was added. The mixturewas stirred for 4 hours and then the resin was capped for 1 h by theaddition of MeOH (258 mL) and DMA

(258 mL). The resin was isolated and washed with DMF (3×3 L). The resincontaining the first amino acid was treated with piperidine in DMF (3×3L of 35%), washed with DMF (9×3 L) and Fmoc-D-Lys(Boc)-OH (472 g) wascoupled using PyBOP (519 g) in the presence of HOBt (153 g) and DMA (516mL) and in DCM/DMF (500 mL/500 mL) with stirring for 2.25 hours. Thedipeptide containing resin was isolated and washed with DMF (3×3.6 L).The Fmoc group was removed by treatment with piperidine in DMF

(3×3.6 L of 35%) and the resin was washed with DMF (9×3.6 L) and treatedwith Fmoc-D-Leu-OH (354 g), DIC (157 mL) and HOBt (154 g) in DCM/DMF(500 mL/500 mL) and stirred for 1 hour. Subsequent washing with DMF(3×4.1 L) followed by cleavage of the Fmoc group with piperidine in DMF(3×4.2 L of 35%) and then washing of the resin with DMF (9×4.2 L)provided the resin bound tripeptide. This material was treated withFmoc-D-Phe-OH (387 g), DIC (157 mL) and HOBt (153 g) in DCM/DMF (500mL/500 mL) and stirred overnight. The resin was isolated, washed withDMF (3×4.7 L) and then treated with piperidine in DMF (3×4.7 L of 35%)to cleave the Fmoc group and then washed again with DMF (9×4.7 L). Thetetrapeptide loaded resin was treated with Fmoc-D-Phe-OH (389 g), DIC(157 mL) and HOBt (154 g) in DCM/DMF (500 mL/500 mL) and stirred for2.25 hours. The resin was isolated, washed with DMF (3×5.2 L) and thentreated piperidine (3×5.2 L of 35%) in DMF. The resin was isolated, andwashed sequentially with DMF (9×5.2 L) then DCM (5×5.2 L). It was driedto provide a 90.4% yield of protected peptide bound to the resin. Thepeptide was cleaved from the resin using TFA/water (4.5 L, 95/5), whichalso served to remove the Boc protecting groups. The mixture wasfiltered, concentrated (⅓) and then precipitated by addition to MTBE (42L). The solid was collected by filtration and dried under reducedpressure to give crude peptide.

For purification, the crude peptide was dissolved in 0.1% TFA in H₂O andpurified by preparative reverse phase HPLC (C18) using 0.1%TFA/water-ACN gradient as the mobile phase. Fractions with purityexceeding 95% were pooled, concentrated and lyophilized to provide purepeptide (>95.5% pure). Ion exchange was conducted using a Dowex ionexchange resin, eluting with water. The aqueous phase was filtered (0.22μm filter capsule) and freeze-dried to give the acetate salt of thepeptide (overall yield, 71.3%, >99% pure).

Example 3 Synthesis of Compound (3)

D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 1):

The synthesis was started with 0.3 mmol of the peptide resin:Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin], which was prepared during the synthesis ofcompound (2) as described below. HPLC and MS analyses were alsoperformed as described in the synthesis of compound (2) above.

For the methylation of the ω-amino function of D-Lys at Xaa₄, athree-step procedure as described in the synthesis of compound (1) wasfollowed. See description above. The resin-bound methylated peptide(Boc-D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin]) was then treated with a mixture of TFA/TIS/H₂O(15 ml, v/v/v=95:2.5:2.5) at room temperature for 90 min. The resin wasthen filtered and washed with TFA. The filtrate was evaporated in vacuoand the crude peptide (0.3 mmol;D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[ω(4-amino-piperidine-4-carboxylicacid)]-OH) was precipitated from diethyl ether.

The crude peptide (0.3 mmol) was purified by preparative HPLC accordingto the protocol described in the synthesis of compound (2). See above.Fractions with purity exceeding 97% were pooled, frozen, and dried on alyophilizer to yield the purified peptide as white amorphous powder (185mg). HPLC analysis: t_(R)=16.93 min, purity 99.2%, gradient 5% B to 25%B over 20 min; MS (M+H⁺): expected molecular ion mass 694.4, observed694.4.

Example 4 Synthesis of Compound (4)

D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]-OH (SEQ ID NO: 2):

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, Fmoc-D-Lys(Dde)-OH, andN-(1-Fmoc-piperidin-4-yl)-L-proline. HPLC and MS analyses were performedas described in the synthesis of compound (1). See detailed descriptionabove. The scheme followed was substantially as shown in FIG. 2, exceptthat couplings were mediated by HATU/DIEA rather than DIC.

The fully protected resin-bound peptide was synthesized manuallystarting from 2-Chlorotrityl chloride resin (3.2 g, 2.4 mmol; NeoMPS).The attachment of the first amino acid to the resin was achieved bytreatment with a mixture of N-(1-Fmoc-piperidin-4-yl)-L-proline (2.0 g,4.8 mmol) and DIEA (3.3 ml, 19.2 mmol) in DCM (40 ml) and DMF (10 ml) atroom temperature for 4 hours. The resin was washed with 3×DCM/MeOH/DIEA(v/v/v=17:2:1) and 3×DCM and dried in vacuo. The resulting resin (3.7 g;N-(4-piperidinyl)-L-proline-[2-Cl-Trt resin]) was split into severalportions and a portion of 1.9 g (1.2 mmol) was used to continue thepeptide synthesis. HATU/DIEA-mediated single couplings were performedwith a 3-fold excess of amino acid derivatives. The Fmoc group wasremoved with 25% piperidine in DMF. Upon completion of peptide chainelongation, the resin was treated with 4% hydrazine in DMF three timesfor 3 min each to remove Dde. The resin was washed with DMF and DCM anddried in vacuo. The resulting peptide resin (2.1 g;Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin]) was split again and a portion of 0.7 g (0.4 mmol) was used forsubsequent cleavage. The peptide resin was treated with a mixture ofTFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for 90 min.The resin was filtered and washed with TFA. The filtrate was evaporatedin vacuo and the crude peptide (220 mg,D-Phe-D-Phe-D-Leu-D-Lys-[N-(4-piperidinyl)-L-proline]-OH) wasprecipitated from diethyl ether.

For purification, the above crude peptide (220 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=43% B. Fractions withpurity exceeding 97% were pooled, frozen, and dried on a lyophilizer togive the purified peptide as white amorphous powder (89 mg). HPLCanalysis: t_(R)=18.22 min, purity 99.5%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 734.5, observed 734.4.

Example 5 Synthesis of Compound (5)

D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]-OH (SEQ ID NO: 3):

The peptide-resin:Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin], which was prepared during the synthesis of compound (4)described above, was used as the starting material. HPLC and MS analyseswere performed as described in the synthesis of compound (1) above.

For guanylation of the ω-amino function of D-Lys at Xaa₄, the peptideresin (0.7 g, 0.4 mmol) was treated with a mixture of1H-Pyrazole-1-carboxamidine hydrochloride (0.6 g, 4.0 mmol) and DMA (0.7ml, 4.0 mmol) in DMF (15 ml) overnight at room temperature. The resinwas washed with DMF and DCM and dried in vacuo. The peptide was thencleaved from the resin by treatment with a mixture of TFA/TIS/H₂O (15ml, v/v/v=95:2.5:2.5) at room temperature for 90 min. The resin was thenfiltered and washed with TFA. The filtrate was evaporated in vacuo andthe crude peptide (170 mg;D-Phe-D-Phe-D-Leu-D-Har-[N-(4-piperidinyl)-L-proline]-OH) wasprecipitated from diethyl ether.

For purification, the above crude peptide (170 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=46% B. Fractions withpurity exceeding 97% were pooled, frozen, and lyophilized to yield thepurified peptide as white amorphous powder (81 mg). HPLC analysis:t_(R)=19.42 min, purity 100%, gradient 5% B to 25% B over 20 min; MS(M+H⁺): expected molecular ion mass 776.5, observed 776.5.

Example 6 Synthesis of Compound (6)

D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]-OH (SEQ IDNO: 1):

Synthesis was initiated with 0.7 g (0.4 mmol) of the peptide resin,Boc-D-Phe-D-Phe-D-Leu-D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin], which was prepared during the synthesis of compound (4) asdescribed above. HPLC and MS analyses were performed as described in thesynthesis of compound (1) above. In this case, the Xaa₁-Xaa₄ peptide waspre-synthesized and coupled as opposed to the stepwise assembly of thepeptide shown in FIG. 2.

For the methylation of the ω-amino function of D-Lys at Xaa₄, athree-step procedure was followed as described in the synthesis ofcompound (1) above. The resin-bound methylated peptide(Boc-D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin]) was then treated with a mixture of TFA/TIS/H₂O (15 ml,v/v/v=95:2.5:2.5) at room temperature for 90 minutes. The resin wasfiltered and washed with TFA. The filtrate was evaporated in vacuo andthe crude peptide (200 mg;D-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]-OH) wasprecipitated from diethyl ether.

For purification, the above crude peptide (200 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof 25% to 75% buffer B, over 30 min, t_(R)=42% B. Fractions with purityexceeding 97% were pooled, frozen, and dried on a lyophilizer to yieldthe purified peptide as white amorphous powder (41 mg). HPLC analysis:t_(R)=18.66 min, purity 98.1%, gradient 5% B to 25% B over 20 min; MS(M+H⁺): expected molecular ion mass 748.5, observed 748.5.

Example 7 Synthesis of Compound (7)

D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide] (SEQ ID NO: 4):

The amino acid derivatives used were Boc-D-Phe-OH, Fmoc-D-Phe-OH,Fmoc-D-Leu-OH, and Fmoc-D-Arg(Pbf)-OH. HPLC and MS analyses wereperformed as in the synthesis of compound (1) described above. The fullyprotected resin bound peptide was synthesized on a SYMPHONY MultipleSynthesizer (Protein Technology Inc.) starting from the homopiperazinecarbamate Wang resin (0.35 mmol; homopiperazine-[carbamate Wang resin])that was prepared during the synthesis of compound (1). HBTU/DIEAmediated single couplings with a 4-fold excess of amino acid derivativeswere performed. The Fmoc group was removed with 25% piperidine in DMF.Upon completion of the automated synthesis, the peptide resin(Boc-D-Phe-D-Phe-D-Leu-D-Arg(Pbf)-[homopiperazine amide]) wastransferred into a manual peptide synthesis vessel and treated with amixture of TFA/TIS/H₂O (15 ml, v/v/v=95:2.5:2.5) at room temperature for90 min. The resin was filtered and washed with TFA. The filtrate wasevaporated in vacuo and the crude peptide (380 mg;D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide]) was precipitated fromdiethyl ether.

For purification, the above crude peptide (380 mg) was dissolved in 0.1%TFA in H₂O (50 ml) and the solution was loaded onto an HPLC column andpurified using a TFA buffer system (buffers A=0.1% TFA in H₂O and B=0.1%TFA in 60% ACN/40% H₂O). The compound was eluted with a linear gradientof buffer B, 25% B to 75% B over 25 min, t_(R)=36% B. Fractions withpurity exceeding 97% were pooled, frozen, and lyophilized to give thepurified peptide as white amorphous powder (222 mg). HPLC analysis:t_(R)=16.75 min, purity 100%, gradient 2% B to 22% B over 20 min; MS(M+H⁺): expected molecular ion mass 664.4, observed 664.5.

Example 8 Synthesis of Compound (8)

D-Phe-D-Phe-D-Leu-D-Har-[ω(4-aminopiperidine-4-carboxylic acid]-OH (SEQID NO: 3):

This compound was prepared essentially according to the proceduredescribed above for the synthesis of compound (5) except thatN-Boc-amino-(4-N-Fmoc-piperidinyl) carboxylic acid was substituted forN-(1-Fmoc-piperidin-4-yl)-L-proline in the attachment to 2-Cl-Trt resin.Final purified peptide: amorphous powder, 85 mg in yield in a synthesisscale of 1 mmol. HPLC analysis: t_(R)=17.87 min, purity 100%, gradient5% B to 25% B over 20 min; MS (M+H⁺): expected molecular ion mass 722.4,observed 722.5.

Example 9 Synthesis of Compound (9)

D-Phe-D-Phe-D-Leu-(ε-iPr)D-Lys-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 5):

Synthesis was initiated from 0.15 mmol of the peptide resin,Boc-D-Phe-D-Phe-D-Leu-D-Lys-(N-Boc-amino-4-piperidinylcarboxylicacid)-[2-Cl-Trt resin]), which was prepared during the synthesis ofcompound (2) above. For isopropylation of the ω-amino function of D-Lysat Xaa₄, the peptide resin was treated with a mixture of sodiumtriacetoxyborohydride (3 mmol) and acetone (6 mmol) in TMOF (10 mL) for4 h at room temperature. Subsequent cleavage and purification steps werecarried out according to the procedure described in the synthesis ofcompound (2). Final purified peptide: amorphous powder, 67 mg in yield.HPLC analysis: t_(R)=19.29 min, purity 98.4%, gradient 5% B to 25% Bover 20 min; MS (M+H⁺): expected molecular ion mass 722.5, observed722.5.

Example 10 Synthesis of Compound (10)

D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]-OH (SEQ ID NO: 6):

See the scheme of FIG. 3. The amino acid derivatives used wereBoc-D-Phe-OH, Boc-D-Phe-OH, Boc-D-Leu-OH, Boc-D-Dap(Fmoc)-OH, andN-Fmoc-amino-(4-N-Boc-piperidinyl) carboxylic acid. HPLC and MS analyseswere performed as described in the synthesis of compound (1). The fullyprotected resin-bound peptide was synthesized manually starting with4-Fmoc-hydrazinobenzoyl AM NovaGel resin (3 mmol; Novabiochem). The Fmocprotecting group on the starting resin was first removed by 25%piperidine in DMF and the resin was then treated with a mixture ofN-Fmoc-amino-(4-N-Boc-piperidinyl) carboxylic acid (7.5 mmol), PyBOP(7.5 mmol), and DIEA (15 mmol) in DMF overnight at room temperature. TheFmoc group on the attached amino acid was replaced by o-NBS in twosteps: (i) Fmoc removal by 25% piperidine in DMF. (ii) o-NBS protectionaccording the procedure described in the synthesis of compound (1). Theresulting peptide resin, N-o-NBS-amino-(4-N-Boc-piperidinyl) carboxylicacid-[hydrazinobenzoyl AM NovaGel resin], was split into severalportions and a portion of 1 mmol was used to continue the peptidesynthesis. PyBOP/DIEA mediated single couplings were performed with a3-fold excess of amino acid derivatives. The Boc group was removed with30% TFA in DCM. Upon completion of peptide chain elongation, the resinwas treated with 2% DBU in DMF for 2×8 min for Fmoc removal, followed byguanylation of the ω-amino function of D-Dap at Xaa₄ according to theprocedure described in the synthesis of compound (5), above. The finalo-NBS deprotection was carried out according to the procedure describedin the synthesis of compound (1).

For oxidative cleavage, the dried peptide resin was mixed with a mixtureof Cu(OAc)₂ (1 mmol), pyridine (4 mmol), and DBU (2 mmol) in 5% H₂O inDMF and let air bubble through the resin for 6 h at room temperature.The resin was filtered and washed with DMF and the filtrated wasevaporated in vacuo. The residue,Boc-D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]-OH, was treated with 95% TFA in H₂O for Boc removal. The solutionwas evaporated in vacuo and the crude peptide (1 mmol;D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[ω(4-aminopiperidine-4-carboxylicacid)]-OH) was precipitated from diethyl ether.

Purification of the above crude peptide was achieved according to theprotocol described in the synthesis of compound (2). The purifiedpeptide was an amorphous powder (16 mg). HPLC analysis: t_(R)=16.97 min,purity 99.9%, gradient 5% B to 25% B over 20 min; MS (M+H⁺): expectedmolecular ion mass 680.4, observed 680.4.

Example 11 Synthesis of Compound (11)

D-Phe-D-Phe-D-Leu-D-Nar-[ω(4-aminopiperidine-4-carboxylic acid)]-OH (SEQID NO: 7):

This compound was prepared according to the procedure described in thesynthesis of compound (10), except that Boc-D-Dbu(Fmoc)-OH wassubstituted for Boc-D-Dap(Fmoc)-OH in the coupling of the amino acidderivative at Xaa₄. Final purified peptide: amorphous powder, 23 mg inyield in a synthesis scale of 1 mmol. HPLC analysis: t_(R)=17.12 min,purity 99.2%, gradient 5% B to 25% B over 20 min; MS (M+H⁺): expectedmolecular ion mass 694.4, observed 694.5.

Example 12 Synthesis of Compound (12)

D-Phe-D-Phe-D-Leu-D-Dbu-[N-(4-piperidinyl)-L-proline]-OH (SEQ ID NO: 8):

This compound was prepared according to the procedure described in thesynthesis of compound (4), as described above. The variation was thesubstitution of Fmoc-D-Dbu(ivDde)-OH for Fmoc-D-Lys(Dde)-OH in thecoupling of the amino acid derivative at Xaa₄. Final purified peptide:amorphous powder, 7 mg in yield in a synthesis scale of 0.4 mmol. HPLCanalysis: t_(R)=18.15 min, purity 98.9%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 706.4, observed 706.4.

Example 13 Synthesis of Compound (13)

D-Phe-D-Phe-D-Leu-D-Nar-[N-(4-piperidinyl)-L-proline]-OH (SEQ ID NO: 7):

Synthesis was initiated from 0.4 mmol of the peptide resin,Boc-D-Phe-D-Phe-D-Leu-D-Dbu-N-(4-piperidinyl)-L-proline-[2-Cl-Trtresin], which was prepared during the synthesis of compound (12). Theguanylation of the ω-amino function of D-Dbu at Xaa₄ was achievedaccording to the procedure described in the synthesis of compound (5),above. Subsequent cleavage and purification steps were carried outaccording to the procedure described in the synthesis of compound (1).Final purified peptide: amorphous powder, 7 mg in yield. HPLC analysis:t_(R)=18.68 min, purity 97.3%, gradient 5% B to 25% B over 20 min; MS(M+H⁺): expected molecular ion mass 748.5, observed 748.5.

Example 14 Synthesis of Compound (14)

D-Phe-D-Phe-D-Leu-D-Dap(amidino)-[N-(4-piperidinyl)-L-proline]-OH (SEQID NO: 6):

The compound was prepared according to the procedure described in thesynthesis of compound (13) except that Fmoc-D-Dap(ivDde)-OH wassubstituted for Fmoc-D-Dbu(ivDde)-OH in the coupling of the amino acidderivative at Xaa₄. Final purified peptide: amorphous powder, 12 mg inyield in a synthesis scale of 0.4 mmol. HPLC analysis: t_(R)=18.55 min,purity 98.0%, gradient 5% B to 25% B over 20 min; MS (M+H⁺): expectedmolecular ion mass 734.4, observed 734.4.

Example 15 Synthesis of Compound (15)

D-Phe-D-Phe-D-Leu-D-Lys-[4-Amidinohomopiperazine amide] (SEQ ID NO: 2):

The compound was prepared according to the procedure described in thesynthesis of compound (1), except that methylation of the ω-aminofunction of D-Lys at Xaa₄ was omitted. Final purified peptide: amorphouspowder, 140 mg in yield in a synthesis scale of 0.3 mmol. HPLC analysis:t_(R)=14.02 min, purity 99.3%, gradient 5% B to 25% B over 20 min; MS(M+H⁺): expected molecular ion mass 678.4, observed 678.5.

Example 16 Synthesis of Compound (16)

D-Phe-D-Phe-D-Leu-D-Har-[4-Amidinohomopiperazine amide] (SEQ ID NO: 3):

The compound was prepared according to the procedure described in thesynthesis of compound (1), except that a guanylation step wassubstituted for the methylation of the ω-amino function of D-Lys atXaa₄. The guanylation was achieved according to the procedure describedin the synthesis of compound (5), above. Final purified peptide:amorphous powder, 173 mg in yield in a synthesis scale of 0.3 mmol. HPLCanalysis: t_(R)=15.05 min, purity 98.6%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 720.5, observed 720.5.

Example 17 Synthesis of Compound (17)

D-Phe-D-Phe-D-Leu-(ε-iPr)D-Lys-[4-Amidinohomopiperazine amide] (SEQ IDNO: 5):

The compound was prepared according to the procedure described in thesynthesis of compound (1) except that an isopropylation step wassubstituted for the methylation of the ω-amino function of D-Lys atXaa₄. The isopropylation was achieved according to the proceduredescribed in the synthesis of compound (9). Final purified peptide:amorphous powder, 233 mg in yield in a synthesis scale of 0.3 mmol. HPLCanalysis: t_(R)=16.16 min, purity 94.5%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 720.5, observed 720.5.

Example 18 Synthesis of Compound (18)

D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[4-Amidinohomopiperazine amide] (SEQID NO: 6):

The compound was prepared according to the procedure described in thesynthesis of compound (16) except for the substitution ofFmoc-D-Dap(ivDde)-OH for Fmoc-D-Lys(Dde)-OH in the coupling of the aminoacid derivative at Xaa₄. Final purified peptide: amorphous powder, 155mg in yield in a synthesis scale of 0.3 mmol. HPLC analysis: t_(R)=14.44min, purity 99.1%, gradient 5% B to 25% B over 20 min; MS (M+H⁺):expected molecular ion mass 678.4, observed 678.5.

Example 19 Synthesis of Compound (19)

D-Phe-D-Phe-D-Nle-(β-amidino)D-Dap-[4-Amidinohomopiperazine amide] (SEQID NO: 6):

The compound was prepared according to the procedure described in thesynthesis of compound (18) above, except for the substitution ofFmoc-D-Nle-OH for Fmoc-D-Leu-OH in the coupling of the amino acidderivative at Xaa₃. Final purified peptide: amorphous powder, 190 mg inyield in a synthesis scale of 0.3 mmol. HPLC analysis: t_(R)=14.69 min,purity 98.9%, gradient 5% B to 25% B over 20 min; MS (M+H⁺): expectedmolecular ion mass 678.2, observed 678.5.

Example 20 Synthesis of Compound (20)

D-Phe-D-Phe-D-Leu-(β-amidino)D-Dap-[homopiperazine amide] (SEQ ID NO:6):

The compound was prepared according to the procedure described in thesynthesis of compound (18) above, except that the guanylation of thehomopiperazine at C-terminus was omitted. Final purified peptide:amorphous powder, 172 mg in yield in a synthesis scale of 0.3 mmol. HPLCanalysis: t_(R)=13.84 min, purity 99.1%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 636.4, observed 636.5.

Example 21 Synthesis of Compound (21)

D-Phe-D-Phe-D-Nle-(β-amidino)D-Dap-[homopiperazine amide] (SEQ ID NO:6):

The compound was prepared according to the procedure described in thesynthesis of compound (19) except that the guanylation of thehomopiperazine at C-terminus was omitted. Final purified peptide:amorphous powder, 149 mg in yield in a synthesis scale of 0.3 mmol. HPLCanalysis: t_(R)=14.06 min, purity 98.5%, gradient 5% B to 25% B over 20min; MS (M+H⁺): expected molecular ion mass 636.4, observed 636.5.

Example 22 Synthesis of Compound (22)

D-Phe-D-Phe-D-Leu-D-Dbu-[4-Amidinohomopiperazine amide] (SEQ ID NO: 8):

The compound was prepared according to the procedure described in thesynthesis of compound (15) except for the substitution ofFmoc-D-Dbu(ivDde)-OH for Fmoc-D-Lys(Dde)-OH in the coupling of the aminoacid derivative at Xaa₄. Final purified peptide: amorphous powder, 152mg in yield in a synthesis scale of 0.3 mmol. HPLC analysis: t_(R)=14.03min, purity 98.1%, gradient 5% B to 25% B over 20 min; MS (M+H⁺):expected molecular ion mass 650.4, observed 650.5.

Example 23 Synthesis of Compound (23)

D-Phe-D-Phe-D-Leu-D-Nar-[4-Amidinohomopiperazine amide] (SEQ ID NO: 7):

The compound was prepared according to the procedure described in thesynthesis of compound (16) except for the substitution ofFmoc-D-Dbu(ivDde)-OH for Fmoc-D-Lys(Dde)-OH in the coupling of the aminoacid derivative at Xaa₄. Final purified peptide: amorphous powder, 227mg in yield in a synthesis scale of 0.3 mmol. HPLC analysis: t_(R)=14.37min, purity 99.3%, gradient 5% B to 25% B over 20 min; MS (M+H⁺):expected molecular ion mass 664.4, observed 664.5.

Example 24 Synthesis of Compound (24)

D-Phe-D-Phe-D-Leu-D-Arg-[4-Amidinohomopiperazine amide] (SEQ ID NO: 4):

The compound was prepared by guanylation of the homopiperazine atC-terminus of Bcz-D-Phe-D-Phe-D-Leu-D-Arg-[homopiperazine amide], whichwas synthesized according to the procedure described in the synthesis ofcompound (7), described above. Subsequent cleavage and purification werecarried out according to the procedure described in the synthesis ofcompound (1), above. Final purified peptide: amorphous powder, 102 mg inyield in a synthesis scale of 0.3 mmol. HPLC analysis: t_(R)=17.34 min,purity 98.4%, gradient 2% B to 22% B over 20 min; MS (M+H⁺): expectedmolecular ion mass 706.5, observed 706.5.

Example 25 Synthesis of Compound (25)

D-Phe-D-Phe-D-Leu-D-Lys-[2,8-diazaspiro[4,5]decan-1-one amide] (SEQ IDNO: 2):

These syntheses were carried out according to the scheme shown in FIG.4. The intermediates described below correspond to those shown in FIG.4. To a suspension of Boc-D-Phe-OH intermediate I-1 (7.96 g, 30.0 mmol),D-Leu-OBn p-TsOH intermediate I-2 (11.80 g, 30.0 mmol), HOBt monohydrate(4.46 g, 33.0 mmol) and DIEA (8.53 g, 66.0 mmol) in anhydrous THF (250mL) cooled in an ice-water bath was added EDCI (6.33 g, 33.0 mmol) infour portions over 20 minutes with 5 minutes between each addition. Thesuspension was stirred overnight from a starting temperature of 0° C. toroom temperature. After evaporation of THF, the residue was dissolved inethyl acetate and washed sequentially with 10% citric acid, saturatedNaHCO₃ and water. The organic phase was dried over sodium sulfate andevaporated under reduced pressure. The residue was dissolved in DCM,passed through a silica gel plug and eluted with 20% ethyl acetate inhexanes. The eluant was evaporated to give the pure product,Boc-D-Phe-D-Leu-OBn, intermediate I-3 (12.40 g, 88%) as a clear oil.LC-MS: m/z=469 (M+H).

Intermediate I-3 (12.40 g, 26.5 mmol) was dissolved in DCM (50 mL). TFA(25 mL) was added and the solution was stirred at room temperature for 2hours. After evaporation of DCM and TFA, the residue was azeotroped withtoluene twice to give the TFA salt of D-Phe-Leu-OBn, intermediate I-4.This crude dipeptide was suspended in THF, to which Boc-D-Phe-OH (6.36g, 24 mmol), HOBt monohydrate (4.04 g, 26.4 mmol) and DIEA (8.7 mL, 50.0mmol) was added at 0° C. EDCI (6.33 g, 6.4 mmol) was added in fourportions over 20 minutes with 5 minutes between each addition. Thesuspension was stirred from 0° C. to room temperature overnight. Afterevaporation of THF, the residue was dissolved in ethyl acetate andwashed sequentially with 10% citric acid, saturated NaHCO₃ and water.The organic phase was dried over sodium sulfate and evaporated underreduced pressure. The residue was recrystallized from 400 mLacetone/hexanes (1:3) to give 9.1 g pure product. The mother liquor wasevaporated and again recrystallized from acetone/hexanes (1:3) to give2.0 g product. The total yield was 11.1 g (68% for two steps). LC-MS:m/z=616 (M+H).

In a flask flushed with nitrogen was added wet palladium on carbon (1.8g) and a solution of Boc-D-Phe-D-Phe-D-Leu-OBn, intermediate I-5 (11.1g, 18.05 mmol) in methanol (50 mL). The mixture was stirred under ahydrogen balloon overnight. After filtration through celite, methanolwas evaporated under reduced pressure. The residue was dissolved inacetone (20 mL) and slowly added to 500 mL water with 25 mL of 1N HClunder vigorous stirring. Pure product Boc-D-Phe-D-Phe-D-Leu-OH,intermediate I-6 was obtained by filtration 9.4 g (99%). LC-MS: m/z=526(M+H).

To a solution of intermediate I-6 (2.06 g, 3.90 mmol), D-Lys(Boc)-OAllhydrochloride (1.26 g, 3.90 mmol) and DIEA (1.7 ml, 9.8 mmol) in DMF wasadded TBTU (1.56 g, 4.88 mmol) in three portions over 15 min at 0° C.After stirring overnight from a starting temperature of 0° C. to roomtemperature, DMF was evaporated under high vacuum. The crude reactionmixture was precipitate in 400 ml ice water and filtered to collect theprecipitate, Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OAll intermediate I-7(2.60 g), which was used without further purification for the next step.

To a solution of intermediate I-7 (2.60 g, 3.3 mmol) in MeCN (75 mL) wasadded pyrrolidine (1.1 ml, 13.3 mmol) and palladiumtetrakis(triphenylphosphine) (400 mg, 0.35 mmol) at 0° C. The reactionmixture was stirred at room temperature for 3 hours and evaporated todryness. The residue was purified by reverse phase column chromatographywith 30% MeCN/water to 90% MeCN/water to give the pure acid,intermediate I-8 (2.0 g, 80%) after evaporation of acetonitrile/water.LC-MS: m/z=754 (M+H).

To a solution of the acid, intermediate I-8 (150 mg, 0.20 mmol), theamine HNR_(a)R_(b), 2,8-diazaspiro [4,5]decan-1-one (57 mg, 0.30 mmol)and DIEA (175 ul, 1.0 mmol) in DMF (5 mL) was added HBTU (11 3 mg, 3.0mmol) at 0° C. After stirring overnight from a starting temperature of0° C. to room temperature, DMF was evaporated under reduced pressure.The residue was stirred with 4N HCl in 1,4-dioxane (2.0 mL) at roomtemperature for 1 hour. After removal of dioxane, the residue wasdissolved in water and purified by reverse phase column chromatographywith a gradient of 10% MeCN/water to 60% MeCN/water in 30 minutes togive pure product, compound (25) (108 mg, 78% yield for the two steps)after evaporation of solvent. LC-MS: m/z=690 (M+H).

Example 26 Synthesis of Compound (26)

D-Phe-D-Phe-D-Leu-D-Lys-[2-methyl-2,8-diazaspiro[4,5]decan-1-one amide](SEQ ID NO: 2):

Compound (26) was prepared essentially as described for compound 25,above except that the amine (HNR_(a)R_(b) in the scheme of FIG. 4) inthe final amide coupling step was2-methyl-2,8-diazaspiro[4,5]decan-1-one in place of 2,8-diazaspiro[4,5]decan-1-one. LC-MS: m/z=704 (M+H).

Example 27 Synthesis of Compound (27)

D-Phe-D-Phe-D-Leu-D-Lys-[1,3,8-triazaspiro[4,5]decane-2,4-dione amide](SEQ ID NO: 2):

Compound (27) was prepared essentially as described for compound 25,above except that the amine 1,3,8-triazaspiro[4,5]decane-2,4-dione wasused in the final step. LC-MS: m/z=705 (M+H).

Example 28 Synthesis of Compound (28)

D-Phe-D-Phe-D-Leu-D-Lys-[5-chloro-1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3)H-oneamide] (SEQ ID NO: 2):

Compound (28) was prepared essentially as described above for compound25, above except that the amine5-chloro-1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3)H-one was used.LC-MS: m/z=394.

Example 29 Synthesis of Compound (29)

D-Phe-D-Phe-D-Leu-D-Lys-[morpholino(piperidin-4-yl)methanone amide] (SEQID NO: 2):

Compound (29) was prepared essentially as described above for compound25, above except that the amine morpholino(piperidin-4-yl)methanone wasused. LC-MS: m/z=366.

Example 30 Synthesis of Compound (30)

D-Phe-D-Phe-D-Leu-D-Lys-[4-phenyl-1-(piperidin-yl-1H-imidazol-2(3H)-oneamide] (SEQ ID NO: 2):

Compound (30) was prepared essentially as described above for compound25, above except that the amine4-phenyl-1-(piperidin-yl-1H-imidazol-2(3H)-one was used. LC-MS: m/z=779(M+H).

Example 31 Synthesis of Compound (31)

D-Phe-D-Phe-D-Leu-D-Lys-[4-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)piperidineamide] (SEQ ID NO: 2):

Compound (31) was prepared essentially as described above for compound25, above except that the amine4-(3,5-dimethyl-4H-1,2,4-triazol-4-yl)piperidine was used. LC-MS:m/z=716 (M+H).

Example 32 Synthesis of Compound (32)

D-Phe-D-Phe-D-Leu-D-Lys-[1-(piperidin-4-yl)indolin-2-one amide] (SEQ IDNO: 2):

Compound (32) was prepared essentially as described above for compound25, above except that the amine 1-(piperidin-4-yl)indolin-2-one wasused.

LC-MS: m/z=752 (M+H).

Example 33 Synthesis of Compound (33)

D-Phe-D-Phe-D-Leu-D-Lys-[1-phenyl-1,3,8-triazaspiro[4.5]decan-4-oneamide] (SEQ ID NO: 2):

Compound (33) was prepared essentially as described above for compound25, above except that the amine1-phenyl-1,3,8-triazaspiro[4.5]decan-4-one was used. LC-MS: m/z=767(M+H).

Example 34 Synthesis of Compound (34)

D-Phe-D-Phe-D-Leu-D-Lys-[imidazo[1,2-a]pyridine-2-ylmethanamine amide](SEQ ID NO: 2):

Compound (34) was prepared essentially as described above for compound25, above except that the amine imidazo[1,2-a]pyridine-2-ylmethanaminewas used. LC-MS: m/z=683 (M+H).

Example 35 Synthesis of Compound (35)

D-Phe-D-Phe-D-Leu-D-Lys-[(5-methylpyrazin-2-yl)methylamine amide] (SEQID NO: 2):

Compound (35) was prepared essentially as described above for compound25, above except that the amine (5-methyl pyrazin-2-yl)methanamine wasused in the final step. LC-MS: m/z=659 (M+H).

Example 36 Synthesis of Compound (36)

D-Phe-D-Phe-D-Leu-D-Lys-[1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-oneamide] (SEQ ID NO: 2):

Compound (36) was prepared essentially as described above for compound25, above except that the amine1-(piperidin-4-yl)-1H-benzo[d]imidazol-2(3H)-one was used. LC-MS:m/z=753 (M+H).

Example 37 Synthesis of Compound (37)

D-Phe-D-Phe-D-Leu-D-Lys-[4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridineamide] (SEQ ID NO: 2):

Compound (37) was prepared essentially as described above for compound25, above except that the amine4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridine was used. LC-MS: m/z=659(M+H).

Example 38 Confirmation of Structures of Synthetic Peptide Amides 1-24

Table I shows the calculated molecular weight of the molecular ion, MH⁺for each compound and the actual molecular weight observed by massspectrometry. Also shown is the type of synthetic phase used in thesynthesis of each compound: solid phase, or mixed; and the type of resinused in the synthesis, whether a 2-chlorotrityl “2-Cl-Trt” resin, ahydrazinobenzoyl “hydrazine” resin, or a p-nitrophenyl-carbonate (Wang)“carbonate” resin. The number of the figure showing the relevantsynthetic scheme for the synthesis of each compound is shown in the lastcolumn.

TABLE I Synthesis and Confirmation of Structures of Compounds (1)-(24)COM- CALC’D OBSERVED SYNTH POUND MH+ MH+ PHASE RESIN FIG. 1 692.5 692.5mixed Carbonate 1 2 680.4 680.3 solid 2-Cl-Trt 2 3 694.4 694.4 solid2-Cl-Trt 2 4 734.5 734.4 solid 2-Cl-Trt 2 5 776.5 776.5 solid 2-Cl-Trt 26 748.5 748.5 solid 2-Cl-Trt 2 7 664.4 664.5 solid Carbonate 1 8 722.4722.5 solid 2-Cl-Trt 2 9 722.5 722.5 solid 2-Cl-Trt 2 10 680.4 680.4solid hydrazine 3 11 694.4 694.5 solid hydrazine 3 12 706.4 706.4 solid2-Cl-Trt 2 13 748.5 748.5 solid 2-Cl-Trt 2 14 734.4 734.4 solid 2-Cl-Trt2 15 678.4 678.5 mixed Carbonate 1 16 720.5 720.5 mixed Carbonate 1 17720.5 720.5 mixed Carbonate 1 18 678.4 678.5 mixed Carbonate 1 19 678.4678.5 mixed Carbonate 1 20 636.4 636.5 solid Carbonate 1 21 636.4 636.5solid Carbonate 1 22 650.4 650.5 mixed Carbonate 1 23 692.4 692.5 mixedCarbonate 1 24 706.5 706.5 mixed Carbonate 1

Example 39 Inhibition of cAMP Production by Stimulation of EndogenousMouse Kappa-Opioid Receptor in R1.G1 Cells

Potency of the synthetic peptide amides as kappa-opioid receptoragonists was determined by measuring the inhibition offorskolin-stimulated adenylate cyclase activity. R1.G1 cells (a mousethymoma cell line that expresses only the kappa-opioid receptor and noother opioid receptor subtype) were first exposed to forskolin (toinduce cAMP) plus the synthetic peptide amide at the test concentration.After incubation, the cAMP level in the challenged R1.G1 cells wasdetermined using a time resolved fluorescence resonance energy transfer(TR-FRET)-based cAMP immunoassay (LANCE™, Perkin Elmer). The detailedmethod is described below:

Mouse R1.G1 cells (ATCC, Manassas, Va.) were grown in suspension in highglucose-DMEM (Dulbecco's Modified Eagle's Medium, Cellgro, Herndon, Va.)containing 10% horse serum and 2% glutaMax (Invitrogen, Carlsbad.Calif.) without added antibiotics. On the day of the experiment, cellswere spun at 1,000 rpm for 5 minutes at room temperature and then washedonce with HBSS (HEPES Buffered Saline Solution, Invitrogen, Carlsbad,Calif.). Cells were then spun again and resuspended in stimulationbuffer (HBSS with 0.05% FAF-BSA (Fatty acid-free bovine serum albumin,Roche Applied Science, Indianapolis, Ind.), 5 mM HEPES) to 2 millioncells per ml. Antibody supplied with the LANCE™ cAMP immunoassay kit wasthen added to the cells according to the manufacturer's instructions,and 12,000 cells per well were then added to the wells containingforskolin to a predetermined fixed final concentration (typically about2.5 uM) and the previously determined amount of the synthetic peptideamide to be tested. The synthetic peptide amides were tested in a rangeof concentrations to determine potency. Cells were incubated with thesynthetic peptide amide plus forskolin for about 20 minutes at roomtemperature. After incubation, cells are lysed by adding 12 ul ofdetection mix as supplied with the LANCE™ kit, followed by incubationfor one hour at room temperature. Time resolved fluorescence was readusing a 330-380 nm excitation filter, a 665 nm emission filter, dichroicmirror 380, and Z=1 mm. A standard curve for cAMP concentration in thisassay permitted determination of the amount of cAMP present in eachwell. A curve was produced by plotting synthetic peptide amideconcentration against cAMP levels in the test cells, and subjected tonon-linear regression using a four-parameter curve fitting algorithm tocalculate the EC₅₀, the concentration of the synthetic peptide amiderequired to produce 50% of the maximal suppression of cAMP production bythe synthetic peptide amide. Table II shows the EC₅₀ values obtained inthis assay with synthetic peptide amide compounds (1) through (36).

TABLE II Kappa Opioid Agonist Activity and Efficacy Com- mKOR hKOR poundEC₅₀ Efficacy EC₅₀ Efficacy No. (nM) (%) (nM) (%)  (1) 0.043 103 0.15 97 (2) 0.048 96 0.16 99  (3) 0.052 96 0.16 100  (4) 0.075 94 0.15 92  (5)0.034 89 0.17 98  (6) 0.036 89 0.13 100  (7) 0.012 98 0.11 100  (8)0.043 90 0.12 96  (9) 0.078 96 0.17 100 (10) 0.826 90 0.30 86 (11) 0.05292 0.15 82 (12) 0.055 89 0.17 100 (13) 0.032 88 0.13 90 (14) 0.349 860.21 81 (15) 0.028 92 0.11 92 (16) 0.021 105 0.11 96 (17) 0.039 103 0.10102 (18) 1.02 103 1.15 93 (19) 2.152 99 nd nd (20) 0.491 102 0.39 84(21) 0.732 103 1.06 99 (22) 0.095 103 0.18 86 (23) 0.091 104 0.17 84(24) 0.036 97 0.09 93 (25) 0.0314 82 0.0027 101 (26) 0.0194 86 0.0083 99(27) 0.0056 87 <0.001 88 (28) 0.0582 83 0.0083 100 (29) 0.0464 86 0.0145100 (30) 0.1293 88 0.0116 95 (31) 0.0216 86 0.0042 95 (32) 0.0485 920.005 102 (33) 0.0767 86 0.0165 101 (34) 0.3539 90 0.0208 101 (35)0.0359 86 0.0064 99 (36) 0.0234 87 0.0052 100 nd—not determined; mKORand hKOR—mouse and human kappa opioid receptors

Example 40 Potency of Synthetic Peptide Amides on the Human Kappa OpioidReceptor

Human Embryonic Kidney cells (HEK-293 cells, ATCC, Manassas, Va.) in 100mm dishes were transfected with transfection reagent, Fugene6 (RocheMolecular Biochemicals) and DNA constructs in a 3.3 to 1 ratio. The DNAconstructs used in the transfection were as follows: (i) an expressionvector for the human kappa opioid receptor, (ii) an expression vectorfor a human chimeric G-protein, and (iii) a luciferase reporterconstruct in which luciferase expression is induced by the calciumsensitive transcription factor NFAT.

The expression vector containing the human kappa opioid receptor wasconstructed as follows: The human OPRK1 gene was cloned from humandorsal root ganglion total RNA by PCR and the gene inserted intoexpression vector pcDNA3 (Invitrogen, Carlsbad, Calif.) to constructhuman OPRK1 mammalian expression vector pcDNA3-hOPRK1.

To construct the human chimeric G-protein expression vector, thechimeric G-protein Gαqi5 was first constructed by replacing the last 5amino acids of human Gαq with the sequence of the last 5 amino acids ofGαi by PCR. A second mutation was introduced to this human Gαqi5 gene atamino acid position 66 to substitute a glycine (G) with an aspartic acid(D) by site-directed mutagenesis. This gene was then subcloned into amammalian expression vector pcDNA5/FRT (Invitrogen) to yield the humanchimeric G-protein expression vector, pcDNA5/FRT-hGNAq-G66D-i5.

To prepare the luciferase reporter gene construct, synthetic responseelements including 3 copies of TRE(12-O-tetradecanoylphorbol-13-acetate-responsive elements) and 3 copiesof NFAT (nuclear factor of activated T-cells) were incorporated upstreamof a c-fos minimal promoter. This response element and promoter cassettewas then inserted into a luciferase reporter gene vector pGL3-basic(Promega) to construct the luciferase reporter gene plasmid constructpGL3b-3TRE-3NFAT-cfos-Luc.

The transfection mixture for each plate of cells included 6 microgramspcDNA3-hOPRK1, 6 micrograms of pcDNA5/FRT-hGNAq-G66D-i5, and 0.6micrograms of pGL3b-3TRE-3NFAT-cfos-Luc. Cells were incubated for oneday at 37° C. in a humidified atmosphere containing 5% CO₂ followingtransfection, and plated in opaque 96-well plates at 45,000 cells perwell in 100 microliters of medium. The next day, test and referencecompounds were added to the cells in individual wells. A range ofconcentrations of test compounds was added to one set of wells and asimilar range of concentrations of reference compounds was added to aset of control wells. The cells were then incubated for 5 hours at 37°C. At the end of the incubation, cells were lysed by adding 100microliters of detection mix containing luciferase substrate (AMP (22ug/ml), ATP (1.1 mg/ml), dithiothreitol (3.85 mg/ml), HEPES (50 mM finalconcentration), EDTA (0.2 mg/ml), Triton N-101 (4 ul/ml), phenylaceticacid (45 ug/ml), oxalic acid (8.5 ug/ml), luciferin (28 ug/ml), pH 7.8).Plates were sealed and luminescence read within 30 minutes. Theconcentration of each of the compounds was plotted against luminescencecounts per second (cps) and the resulting response curves subjected tonon-linear regression using a four-parameter curve-fitting algorithm tocalculate the EC₅₀ (the concentration of compound required to produce50% of the maximal increase in luciferase activity) and the efficacy(the percent maximal activation compared to full induction by any of thewell-known kappa opioid receptor agonists, such as asimadoline(EMD-61753: See Joshi et al., 2000, J. Neurosci. 20(15):5874-9), orU-69593: See Heidbreder et al., 1999, Brain Res. 616(1-2):335-8).

Table II shows the EC₅₀ values obtained from the cAMP inhibition assaywith the exemplified compounds synthesized according to the presentinvention and tested on mouse kappa opioid receptor (mKOR) withconfirmatory replication of results on the human kappa opioid receptor(hKOR) by the above-described methods.

Synthetic peptide amides of the invention were tested in a similar assayfor potency on the human mu opioid receptor. Each compound tested had anEC₅₀ for the human mu opioid receptor greater than or equal to 1 uM.

Example 41 Membrane Permeability of the Synthetic Peptide Amides

The Caco-2 cell line is a human colon adenocarcinoma cell line thatdifferentiates in culture and is used to model the epithelial lining ofthe human small intestine. Compounds of the present invention weretested in a membrane permeability assay using the TC7 subclone of Caco-2in a standard assay (Cerep, Seattle, Wash.). Briefly, the apparentpermeability coefficient (P_(app)) was determined in theapical-to-basolateral (A-B) direction across cell monolayers cultured on96-well polycarbonate membrane filters.

Compounds were tested at a concentration of 10 uM at pH 6.5 in 1% DMSO,with the recipient side maintained at pH 7.4. The assay plate wasincubated for 60 minutes at 37° C. with gentle shaking. Samples weretaken at time zero from the donor side and at the end of the incubationperiod from both the donor and recipient sides. Samples were analyzed byHPLC-MS/MS. The P_(app)-value (expressed as 10⁻⁶ cm/sec) was thencalculated based on the appearance rate of compound in the recipientside. The P_(app) was calculated with the equation:

$P_{app} = {\frac{1}{S \cdot C_{0}}\left( \frac{Q}{T} \right)}$

where P_(app) is the apparent permeability; S is the membrane surfacearea, C₀ is the donor concentration at time 0, and dQ/dt is the amountof drug transported per time. Four reference compounds (labetalol,propranolol, ranitidine, and vinblastine) were concurrently tested toensure the validity of the assay, as well as asimadoline, which ispurported to be a peripherally acting kappa opioid. Results are shown inTable III.

TABLE III Membrane permeability Com- Mean Permeability pound (cm⁻⁶/sec)(1) <0.10 (3) <0.02 (6) <0.02 Asimadoline 37.5 Labetalol 9.9 Propranolol53.8 Ranitidine 0.5 Vinblastine <0.2

Compounds that exhibit low permeability in this type of assay arebelieved to have reduced potential for crossing the blood-brain barrierin vivo, since high passive permeability appears to be a key feature ofCNS-acting drugs (Mahar Doan et al. Passive permeability andP-glycoprotein-mediated efflux differentiate central nervous system(CNS) and non-CNS marketed drugs. J Pharmacol Exp Ther. 2002;303:1029-37).

Example 42 Inhibition of Cytochrome P₄₅₀ Oxidases

Inhibition of cytochrome P₄₅₀ oxidase isozymes CYP1A, CYP2C9, CYP2C19,CYP2D6 and CYP3A4 by synthetic peptide amide compounds of the inventionwas determined according to the following methods performed by Cerep(Seattle, Wash.):

In the cytochrome P₄₅₀ CYP1A assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 15 minutes at 37° C. with 10 uM testcompound, 1 uM ethoxyresorufin, 1.3 mM NADP, 3.3 mM glucose-6-phosphateand 0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the ethoxyresorufin added as substrate is oxidized toresorufin, and in the presence of an inhibitor of the CYP isozyme, theamount of resorufin produced is reduced. Furafylline was used as areference inhibitor.

The cytochrome P₄₅₀ CYP2C9 assay reaction mixture containing human livermicrosomes (0.2 mg/ml protein) was incubated for 15 minutes at 37° C.with 10 uM test compound, 10 uM tolbutamide, 1.3 mM NADP, 3.3 mMglucose-6-phosphate and 0.4 U/ml glucose-6-phosphate dehydrogenase. Inthe absence of test compound, the tolbutamide is oxidized to4-hydroxytolbutamide, and in the presence of an inhibitor of the CYPisozyme, the amount of 4-hydroxytolbutamide produced is reduced.Sulfaphenazole (IC₅₀: 0.35 uM) was the reference inhibitor.

For the cytochrome P₄₅₀ CYP2C19 assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 15 minutes at 37° C. with 10 uM testcompound, 10 uM omeprazole, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the omeprazole is oxidized to 5-hydroxy-omeprazole, and in thepresence of an inhibitor of the CYP isozyme, the amount of5-hydroxy-omeprazole produced is reduced. Oxybutinin (IC₅₀: 7.1 uM) wasthe reference inhibitor.

The cytochrome P₄₅₀ CYP2D6 assay reaction containing human livermicrosomes (0.2 mg/ml protein) was incubated for 15 minutes at 37° C.with 10 uM test compound, 5 uM dextromethorphan, 1.3 mM NADP, 3.3 mMglucose-6-phosphate and 0.4 U/ml glucose-6-phosphate dehydrogenase. Inthe absence of test compound, the dextromethorphan is oxidized, and inthe presence of an inhibitor of the CYP isozyme, the amount of oxidationproduct is reduced. Quinidine (IC₅₀: 0.093 uM) was the referenceinhibitor.

For the cytochrome P₄₅₀ CYP2C19 assay, human liver microsomes (0.2 mg/mlprotein) were incubated for 20 minutes at 37° C. with 10 uM testcompound, 5 uM midazolam, 1.3 mM NADP, 3.3 mM glucose-6-phosphate and0.4 U/ml glucose-6-phosphate dehydrogenase. In the absence of testcompound, the midazolam is oxidized, and in the presence of an inhibitorof the recombinant isozyme, the amount of oxidation product is reduced.The oxidation product is determined from the area under the curve afterHPLC-MS/MS separation. Ketoconazole (IC₅₀: 0.55 uM) was the referenceinhibitor.

In each assay, the percent inhibition of the cytochrome P₄₅₀ CYP P₄₅₀isozyme was determined as one hundred times the ratio of (1−the amountof product in the sample in the presence of the test compound) dividedby the amount of product in the sample containing untreated isozyme. Theresults of duplicate assays (expressed as percent remaining CYPactivity) are shown in Table IV.

TABLE IV Percent Activity of Cytochrome P₄₅₀ CYP Isozymes Compound P₄₅₀isozyme (1) (3) (6) CYP1A 89.8 93.1 89.5 CYP2C9 93.2 97.4 92.1 CYP2C1998.5 103.2 97.2 CYP2D6 96.0 99.5 93.9 CYP3A4 92.5 94.3 93.6

Example 43 Stability of Compound (2) to Human Liver Microsomes

Microsomes from human liver (final concentration 0.3 mg/mL protein) in0.1M phosphate buffer pH7.4 and NADPH regenerating system (1 mM NADP, 5mM glucose-6-phosphate, and 1 Unit/mL glucose-6-phosphate dehydrogenase)were pre-incubated at 37° C. prior to the addition of substrate,compound (2) to give a final substrate concentration of 1 μM; and afinal methanol concentration of 0.6%. Aliquots were removed at 0 and 60minutes. An equal volume of 50/50 acetonitrile/methanol was added,samples were centrifuged, and the amount of compound remaining in thesupernatant was measured via HPLC coupled with tandem mass spectrometryby comparison of peak areas generated from the 0 and 60 minute samples.Duplicate samples showed 96% and 118% of compound (2) remaining after 60minutes incubation with human liver microsomes.

Example 44 Pharmacokinetics of Compound (2) in Rat

To determine brain to plasma concentration ratios of compound (2), agroup of 6 conscious jugular vein catheterized rats were administered 3mg/kg of peptide over a 5 minute infusion period into the jugular veincatheter. Thirty, 60 and 180 minutes following the start of infusion,blood samples were collected from 2 animals at each time point byterminal cardiac puncture and whole brains were rapidly removed. Plasmawas isolated by centrifugation. Tandem liquid chromatography massspectrometry (LC-MS/MS) was used to quantify the concentration of drugin rat plasma and brain. Results are shown in FIG. 5.

Example 45 Pharmacokinetics of Compound (6) in Mice and CynomolgusMonkeys

A single bolus of the synthetic peptide amide compound was administeredby subcutaneous injection to ICR mice (n=6, males, body wt 23-37 g,Charles River, Wilmington, Mass.) and plasma samples taken at 5, 10, 15,20, 30 60, 90, 120, and 180 minutes post-injection. FIG. 6 shows theresults obtained after subcutaneous injection of a 1 mg/kg dose ofcompound (6) in ICR mice. The “half life” for this study was determinedas the time required for the plasma concentration to fall by 50% aftermaximum concentration in the plasma was achieved; the computedelimination half-life, based on the elimination rate constant of theslowest elimination phase, is expected to be longer. See Table V below.

Example 46 Synthetic Peptide Amide Compound Pharmacokinetics in Monkeys

Samples were administered to male monkeys, Macaca fascicularis (SNBLUSA, Ltd., Everett, Wash., purpose-bred cynomolgus monkeys, closelyrelated to humans, both phylogenetically and physiologically), aged 3-7years and weighing 3-5 kilograms. Samples were administered in asuperficial vein of the arm or leg (e.g. brachial, or saphenous) in 0.9%saline for injection, USP (Baxter Healthcare, Deerfield, Ill.) asfollows: A sample containing 4 mg of compound (6) of the presentinvention to be tested was prepared in 2 ml 0.9% saline for injection.The 2 ml dose was administered as an intravenous bolus to the testanimal, resulting in a dose of approximately 0.4 to 0.65 mg/kg,depending on the body weight of the animal. Blood samples of 0.6 ml werecollected by venipuncture from a peripheral vein at 2, 5, 10, 15 and 30minutes post dose injection, and then at 1, 2 and 4 hours. Each samplewas placed in a pre-chilled glass test tube containing lithium heparinand immediately chilled on ice. Plasma was collected aftercentrifugation at 2,000 g for fifteen minutes at 2-8° C. The plasmalayers of each sample were transferred to polypropylene tubes and storedfrozen at −60° C. or lower until assayed.

One hundred microliter aliquots of thawed plasma were spiked with 5microliters of a 400 ng/ml solution of an appropriate internal standard(in this case a known standard synthetic peptide amide compound) in 0.1%TFA, and the proteins were precipitated with 100 microliters of 0.1% TFAin acetonitrile. The samples were centrifuged at 1000×g for 5 minutesand the supernatants analyzed by LC-MS. LC-MS analysis was performed ona Finnigan LCQ Deca mass spectrometer interfaced to a Surveyor HPLCsystem (Thermo Electron Corporation, Waltham, Mass., USA). HPLC analysiswas performed on 2.1×150 mm C18 reversed phase columns with a gradientof 0.01% TFA in acetonitrile in 0.01% TFA in water. Mass detection wasperformed in the selected reaction monitoring mode (SRM).

Quantitation was performed against a calibration curve of the analyte inblank Cynomolgus monkey plasma using the same internal standard. Dataanalysis and the extraction of pharmacokinetic parameters were performedwith the program PK Solutions 2.0 (Summit Research Services, Ashland,Ohio, USA). Table V, below shows the half life in ICR mice aftersubcutaneous injection of compound (6) and the half life for thiscompound after intravenous bolus administration in cynomolgus monkeys.

TABLE V In vivo half life of synthetic peptide amide compound (6) ICRMice Cynomolgus Monkeys Administration Route subcutaneous intravenousHalf Life (min) 22.0 58.6 *Compound (6) isD-Phe-D-Phe-D-Leu-(ε-Me)D-Lys-[N-(4-piperidinyl)-L-proline]-OH (SEQ IDNO: 1).

Persistence of compound (3) in the plasma of cynomolgus monkeys afterintravenous administration of a bolus of 0.56 mg/kg is shown in FIG. 7.

Example 47 Acetic Acid-Induced Writhing Assay in Mice

This test identifies compounds which exhibit analgesic activity againstvisceral pain or pain associated with activation of low pH-sensitivenociceptors [see Barber and Gottschlich (1986) Med. Res. Rev. 12:525-562; Ramabadran and Bansinath (1986) Pharm. Res. 3: 263-270].Intraperitoneal administration of dilute acetic acid solution causes awrithing behavior in mice. A writhe is defined as a contraction of theabdominal muscles accompanied by an extension of the forelimbs andelongation of the body. The number of writhes observed in the presenceand absence of test compounds is counted to determine the analgesicactivity of the compounds.

Each day a writhing assay was performed, a vehicle control group of mice(n=6-8) that were treated identically to the test group (except thattest compound was omitted from the injection dose) was always includedand the average total number of writhes in this group used as theabsolute reference point defining 0% decrease in pain perception for allother mice receiving a test compound on that day. Specifically, thetotal number of writhes of each mouse receiving the test compound wasconverted to % decrease in pain perception according to the followingequation:

${\% \mspace{14mu} {decrease}\mspace{14mu} {in}\mspace{14mu} {pain}\mspace{14mu} {perception}} = {\frac{\left( {W_{v} - W_{c}} \right)}{W_{v}} \times 100}$

Where W_(v) is the mean number of writhes in vehicle-treated group andW_(c) is the number of writhes in compound-treated mouse. The data wereanalyzed using the 2-parameter Hill's equation (also known as the Emaxmodel), where Emax is assumed to be 100% antinoci-perception (i.e., nowrithes over the 15 min post-acetic acid administration).

Male ICR mice, 23-37 grams in weight, were weighed and placed inindividual observation chambers (usually a 4000 ml glass beaker) with afine layer of SANI-CHIPS rodent bedding at the bottom. To determine theactivity and potency of test compounds, different doses of the compoundsolution or vehicle were injected subcutaneously in the back of the neck15 or 180 minutes prior to administration of acetic acid solution. Afteradministration of the compound or vehicle control, mice were returned totheir individual observation chambers awaiting the intraperitonealadministration of acetic acid solution. Fifteen minutes or three hourslater, according to the interval time defined in each experiment betweencompound delivery and acetic acid injection, a dose corresponding to 10ml/kg of a 0.6% (v/v) acetic acid solution was then injected into theright lower quadrant of the abdomen. Immediately after the injection,the mouse was returned to its observation chamber and the recording ofthe number of writhes begun immediately. The number of writhes wascounted over a 15-min period starting from the time of acetic acidinjection, the data being collected over three separate 5 minute timeperiods (0-5 min, 5-10 min, and 10-15 min).

The data were reported as ED₅₀, and Hill coefficient. The ED₅₀ isexpressed either as mean±standard error of the mean (sem) (ED₅₀+/−sem)or as geometric mean with 95% confidence intervals (95% CI) usingt-scores. The Hill coefficient is expressed as the arithmetic mean±semcalculated from the values obtained from the animals. Results forcompound (2) are shown in FIG. 8 (solid circles).

For dose-response analysis, raw data were converted to % maximumpossible effect (% MPE) using the formula: % MPE=((testscore−vehicle-treated score)/(0−vehicle-treated score))*100. Raw datawere analyzed using a one-way ANOVA followed by Dunnett's post-tests.The dose which elicited 50% attenuation of hypersensitivity (ED₅₀) wasdetermined using linear regression analysis. Compounds were administeredby the intravenous route. Table VI summarizes the results of theseexperiments.

TABLE VI Effects of Compounds (2) and (5) on Acetic Acid-InducedWrithing in Mice. ED₅₀ % MPE % MPE % MPE Com- (mg/kg, iv, 15 min (180min (240 min (300 min pound post-dose) post-ED₉₀) post-ED₉₀) post-ED₉₀)(2) 0.07 77 ± 5%  81 ± 4% 84 ± 4% (0.06-0.1)  (5) 0.01 54 ± 10% NT NT(0.01-0.02) NT = not tested

A dose response for compound (2) in the acetic acid-induced writhingmodel in mice was generated using 0.01, 0.03, 0.1 and 0.3 mg/kgadministered intravenously as described above. Using the above method alinear dose response relationship was determined for compound (2) fordoses ranging from 0.01 mg/kg to 0.3 mg/kg, as shown in FIG. 9.

Example 48 Inhibition of Locomotion in Mice to Measure Sedation byCompounds after Subcutaneous Injection (Locomotion Reduction Assay)

Compounds which exhibit sedative activity inhibit the spontaneouslocomotion of mice in a test chamber. To determine the potentialsedative effect of test compounds, the extent of locomotion reductionafter the administration of the test compound or vehicle control can bedetermined and compared with a specialized apparatus designed for thispurpose (Opto-Varimex Activity Meter). At the start of each experiment,each mouse was weighed and examined to determine good health. Todetermine the activity and potency of compounds, different doses of thecompound solution or vehicle were injected subcutaneously 15 or 180minutes prior to initiation of data collection. The subcutaneousinjection was performed in the back of the neck of the mouse, pinched ina “tent” to allow proper access for the syringe needle. After injection,each animal was placed individually in Plexiglas boxes (43 cm×43 cm)inside the Opto-Varimex Activity Meter apparatus. Before the animal wasplaced in the apparatus, a thin layer of SANI-CHIPS rodent bedding wasplaced on the bottom of the Plexiglas box to provide a comfortableenvironment. Each Opto-Varimex Activity Meter apparatus was then turnedon and data acquisition begun by the ATM3 Auto-Track System. The datawere processed and results expressed in the same way as described forthe writhing assay data in Example 47.

Example 49 Analgesic Effect Vs. Sedative Effect of Synthetic PeptideAmide (3)

Inhibition of acetic acid-induced writhing by a compound is anindication of an analgesic effect (also called an antinociceptiveeffect). Similarly, a reduction in locomotion caused by administrationof the compound can be used as a measure of its general sedative effect.

The ED₅₀ determined in the acetic acid-induced writhing assay in ICRmice was 52 ug/kg when synthetic peptide amide (3) was administeredsubcutaneously as described in Example 34 and shown in FIG. 8 (solidcircles). The ED₅₀ value determined in the inhibition of locomotionassay as described in Example 35 was 2685 ug/kg for the same syntheticpeptide amide administered subcutaneously. See FIG. 8 (solid squares).The therapeutic ratio of the analgesic effect over the sedative effectis the fold higher ED₅₀ required to achieve a sedative effect ascompared to the ED₅₀ required to achieve an analgesic effect. Thus,compound (3) exhibits a (2685/52) fold ratio, i.e. 51.6 fold. Thus, thetherapeutic ratio is approximately 52 fold for compound (3).

Example 50 Spinal Nerve Ligation (SNL) Model

The SNL model (Kim and Chung 1992) was used to induce chronicneuropathic pain. The rats were anesthetized with isoflurane, the leftL5 transverse process was removed, and the L5 and L6 spinal nerves weretightly ligated with 6-0 silk suture. The wound was then closed withinternal sutures and external staples. Fourteen days following SNL,baseline, post-injury and post-treatment values for non-noxiousmechanical sensitivity were evaluated using 8 Semmes-Weinstein filaments(Stoelting, Wood Dale, Ill., USA) with varying stiffness (0.4, 0.7, 1.2,2.0, 3.6, 5.5, 8.5, and 15 g) according to the up-down method (Chaplanet al. 1994). Animals were placed on a perforated metallic platform andallowed to acclimate to their surroundings for a minimum of 30 minutesbefore testing. The mean and standard error of the mean (SEM) weredetermined for each paw in each treatment group. Since this stimulus isnormally not considered painful, significant injury-induced increases inresponsiveness in this test are interpreted as a measure of mechanicalallodynia. The dose which elicited 50% attenuation of mechanicalhypersensitivity (ED₅₀) was determined using linear regression analysis.Compound (2) was administered by the intravenous route. FIG. 10summarizes the results of these experiments. The calculated ED₅₀ forcompound (2) in this model was 0.38 mg/kg (0.31-0.45; 95% confidenceinterval).

Example 51 Ocular Analgesia Induced by Compounds (2), (3) and (4)

Ocular analgesia was evaluated by instilling five volumes of the testcompound, 50 microliters each in physiological saline, at theconcentration to be tested into the right eye of naïve albino NewZealand strain rabbits within a period of twenty minutes. Fifteenminutes after the last instillation of the test compound, each animalwas administered a single instillation of 30 microliters of 10 mg/mlcapsaicin (33 mM) in the treated eye. Capsaicin is known to inducecorneal pain. Corneal pain was evaluated by measurement of the palpebralopening measured in millimeters using a transparent ruler over thetreated and untreated eyes. In this animal model, the reduction in sizeof the palpebral opening after instillation of capsaicin is anindication of the degree of ocular pain. Thus, any observed restoration(increase) in size of the palpebral opening after treatment with testcompound is taken as a measure of relief from capsaicin-induced ocularpain.

These evaluations were performed before treatment with the test compound(pre-test), immediately prior to the instillation of capsaicin, and then1, 5, 10, 15, 20, 25, 30, 40, 50 and 60 minutes following theinstillation of capsaicin. Table VII shows the mean of palpebral openingmeasurements (relative to the untreated eye expressed as percent ofcontrol) averaged over the period from 10-30 minutes after capsaicininstillation in rabbits pre-instilled with a kappa opioid agonist of theinvention and after preinstillation with a standard concentration ofdiltiazem, a benzothiazepine calcium channel blocker with localanesthetic effects. See Gonzalez et al., (1993) Invest. Ophthalmol. Vis.Sci. 34: 3329-3335.

TABLE VII Effect of compounds (2), (3) and (4) in reducing ocular painCom- Time Mean SEM pound (post capsaicin) (% Control) (% Control) None(Saline) 10-30 min. 61.2 6.5 Diltiazem at 10 mM 10-30 min. 74.6 5.5 (2)at 10 mg/ml 10-30 min. 82.7 5.3 (3) at 10 mg/ml 10-30 min. 76.0 5.2 (4)at 10 mg/ml 10-30 min. 56.7 8.4 Mean is of five animals; SEM: Standarderror of the mean

Example 52 Dose Response of Compound (2) in Capsaicin-Induced OcularPain

Ocular analgesia induced by Compound (2) at several concentrationsinstilled into the right eye of naïve albino New Zealand strain rabbitswas evaluated as described above. Results were compared with analgesiainduced by 10 mg/ml morphine (a non-selective opioid agonist) as asystemic active control, and with 10 mM diltiazem as a topicalactive-control in the same experiment and under the same conditions.Table VIII below shows the accumulated results.

TABLE VIII Dose-Response of Compound (2) in Capsaicin-Induced OcularPain Com- Time Mean SEM pound (post capsaicin) (% Control) (% Control)Morphine at 10 mg/ml 10-30 min. 74.8 11.1 Diltiazem at 10 mM 10-30 min.77.6 7.4 (2) at 1 mg/ml  10-30 min. 60.5 9.9 (2) at 10 mg/ml 10-30 min.56.6 9.3 (2) at 25 mg/ml 10-30 min. 75.5 7.1 (2) at 50 mg/ml 10-30 min.87.5 4.8 Mean is of ten animals; SEM: Standard error about the mean

Example 53 Effect of Compound (2) in a Rat Pancreatitis Model

Chronic pancreatic inflammation was induced in rats by intravenousadministration of dibutylin dichloride (DBTC, Aldrich Milwaukee, Wis.)dissolved in 100% ethanol at a dose of 8 mg/kg under isofluoraneanesthesia (2-3 liters/min, 4%/vol until anesthetized, then 2.5%/volthroughout the procedure. Control animals received the same volume ofvehicle (100% ethanol) alone. Pancreatitis pain was assessed bydetermination of abdominal sensitivity to probing the abdomen of ratswith a calibrated von Frey filament (4 g). Rats were allowed toacclimate in suspended wire-mesh cages for 30 min before testing. Aresponse was indicated by the sharp withdrawal of the abdomen, lickingof abdominal area, or whole body withdrawal. A single trial consisted of10 applications of von Frey filament applied once every 10 s to allowthe animal to cease any response and return to a relatively inactiveposition. The mean occurrence of withdrawal events in each trial isexpressed as the number of responses to 10 applications. Rats withoutinflammation of the pancreas typically display withdrawal frequencies toprobing with von Frey filament of 0-1. The animals were allowed torecover for 6 days after DBTC administration prior to anypharmacological manipulations. Animal not demonstrating sufficientabdominal hypersensitivity (i.e., rats with less than 5 positiveresponses out of a possible 10) were excluded from the study.

The number of positive responses, following abdominal probing (out of apossible 10), were recorded at each time point. Data are presented asaverage number of withdrawals (±SEM) for each dosing group at eachcorresponding time point. For dose-response analysis, raw data wereconverted to % maximum possible effect (% MPE) using the formula: %MPE=((test score−post DBTC score)/(pre DBTC score−post DBTC score))*100.Raw data were analyzed using a two-way repeated measures ANOVA followedby Bonferroni post-tests. The dose which elicited 50% attenuation ofhypersensitivity (ED₅₀) was determined using linear regression analysis.Compounds were administered by the intraperitoneal route. FIG. 11summarizes the results of these experiments. The calculated ED₅₀ forcompound (2) in this model was 0.03 mg/kg (0.006-0.14; 95% confidenceinterval).

To determine if the efficacy of Compound (2) (1 mg/kg) is mediated viaactivation of peripheral kappa opioid receptors, groups of eight ratswere pretreated with either the selective kappa opioid receptorantagonist nor-BNI (1 mg/kg), or with a non-selective opioid receptorantagonist, naloxone methiodide (10 mg/kg), which does not cross theblood-brain barrier, prior to treatment with compound (2). FIG. 12summarizes the results of these studies.

Example 54 Pruritus Model in Mice

Groups of 10 (and in one case, 11) male Swiss Webster mice (25-30 g)were used. Each animal was weighed and allowed to acclimate for at leastone hour in individual, rectangular observation boxes. The tails of micewere immersed for 30 seconds in warm water to dilate tail veins and theanimals then received an intravenous injection of either vehicle(saline) or compound (2) (0.01, 0.03, 0.10 and 0.30 mg of free base/kg).Fifteen minutes later, each mouse was given either GNTI dihydrochloride(Tocris) (0.30 mg/kg; 0.25 ml/25 g) or compound 48/80 (Sigma) (50 μg in0.10 ml saline) subcutaneously behind the neck. The animals were thenobserved in pairs (occasionally in threes) and the number of hind legscratching movements directed at the neck was counted for 30 minutes.The mean percent inhibition of scratching caused by compound (2) wasplotted and the dose associated with 50% inhibition was obtained bylinear regression analysis (PharmProTools). Table IX summarizes theresults of these experiments.

TABLE IX Effects of Compound (2) on Pruritus Induced by Either Compound48/80 or GNTI in Mice. Com- Compound 48/80 Model ED₅₀ GNTI Model ED₅₀pound (mg/kg, iv, 15 min (mg/kg, iv, 15 min # post-dose) post-dose) (2)0.08 (0.04-0.2) 0.05 (0.02-0.1)

The specifications of each of the U.S. patents and published patentapplications, and the texts of the literature references cited in thisspecification are herein incorporated by reference in their entireties.In the event that any definition or description contained found in oneor more of these references is in conflict with the correspondingdefinition or description herein, then the definition or descriptiondisclosed herein is intended.

The examples provided herein are for illustration purposes only and arenot intended to limit the scope of the invention, the full breadth ofwhich will be readily recognized by those of skill in the art.

What is claimed is:
 1. A synthetic peptide amide having the formula:

or a stereoisomer, mixture of stereoisomers, prodrug, pharmaceuticallyacceptable salt, hydrate, solvate, acid salt hydrate, N-oxide orisomorphic crystalline form thereof; wherein Xaa₁ is selected from thegroup consisting of (A)(A′)D-Phe, (A)(A′)(α-Me)D-Phe, D-Tyr, D-Tic,D-tert-leucine, D-neopentylglycine, D-phenylglycine,D-homophenylalanine, and β-(E)D-Ala, wherein each (A) and each (A′) arephenyl ring substituents independently selected from the groupconsisting of —H, —F, —NO₂, —CH₃, —CF₃, —CN, and —CONH₂, and whereineach (E) is independently selected from the group consisting ofcyclobutyl, cyclopentyl, cyclohexyl, pyridyl, thienyl and thiazolyl;Xaa₂ is selected from the group consisting of (A)(A′)D-Phe,3,4-dichloro-D-Phe, (A)(A′)(α-Me)D-Phe, D-1Nal, D-2Nal, D-Tyr, (E)D-Alaand D-Trp; Xaa₃ is selected from the group consisting of D-Nle, D-Phe,(E)D-Ala, D-Leu, (α-Me)D-Leu, D-Hle, D-Val, and D-Met; Xaa₄ is selectedfrom the group consisting of (B)₂D-Arg, (B)₂D-Nar, (B)₂D-Har,ζ-(B)D-Hlys, D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf,γ-(B)₂D-Dbu, δ-(B)₂α-(B′)D-Orn, D-2-amino-3(4-piperidyl)propionic acid,D-2-amino-3(2-aminopyrrolidyl)propionic acid,D-α-amino-β-amidinopropionic acid, α-amino-4-piperidineacetic acid,cis-α,4-diaminocyclohexane acetic acid,trans-α,4-diaminocyclohexaneacetic acid,cis-α-amino-4-methylaminocyclo-hexane acetic acid,trans-α-amino-4-methylaminocyclohexane acetic acid,α-amino-1-amidino-4-piperidineacetic acid,cis-α-amino-4-guanidinocyclohexane acetic acid, andtrans-α-amino-4-guanidinocyclohexane acetic acid, wherein each (B) isindependently selected from the group consisting of H and C₁-C₄ alkyl,and (B′) is H or (α-Me); W is selected from the group consisting of:Null, provided that when W is null, Y is N; —NH—(CH₂)_(b)— with b equalto zero, 1, 2, 3, 4, 5, or 6; and —NH—(CH₂)_(c)—O— with c equal to 2, or3, provided that Y is C; the moiety

is an optionally substituted 4 to 8-membered heterocyclic ring moietywherein all ring heteroatoms in said ring moiety are N; wherein Y and Zare each independently C or N; provided that when such ring moiety is asix, seven or eight-membered ring, Y and Z are separated by at least tworing atoms; and provided that when such ring moiety has a single ringheteroatom which is N, then such ring moiety is non-aromatic; V is C₁-C₆alkyl, and e is zero or 1, wherein when e is zero, then V is null and R₁and R₂ are directly bonded to the same or different ring atoms; wherein(i) R₁ is selected from the group consisting of —H, —OH, halo, —CF₃,—NH₂, —COOH, C₁-C₆ alkyl, C₁-C₆ alkoxy, amidino, C₁-C₆ alkyl-substitutedamidino, aryl, optionally substituted heterocyclyl, Pro-amide, Pro, Gly,Ala, Val, Leu, Ile, Lys, Arg, Orn, Ser, Thr, —CN, —CONH₂, —COR′, —SO₂R′,—CONR′R″, —NHCOR′, OR′ and SO₂NR′R″; wherein said optionally substitutedheterocyclyl is optionally singly or doubly substituted withsubstituents independently selected from the group consisting of C₁-C₆alkyl, C₁-C₆ alkoxy, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, andamidino; wherein R′ and R″ are each independently —H, C₁-C₈ alkyl, aryl,or heterocyclyl or R′ and R″ are combined to form a 4- to 8-memberedring, which ring is optionally singly or doubly substituted withsubstituents independently selected from the group consisting of C₁-C₆alkyl, —C₁-C₆ alkoxy, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH and amidino;and R₂ is selected from the group consisting of —H, amidino, singly ordoubly C₁-C₆ alkyl-substituted amidino, —CN, —CONH₂, —CONR′R″, —NHCOR′,—SO₂NR′R″ and —COOH; or (ii) R₁ and R₂ taken together can form anoptionally substituted 4- to 9-membered heterocyclic monocyclic orbicyclic ring moiety which is bonded to a single ring atom of the Y andZ-containing ring moiety; or (iii) R₁ and R₂ taken together with asingle ring atom of the Y and Z-containing ring moiety can form anoptionally substituted 4- to 8-membered heterocyclic ring moiety to forma spiro structure; or (iv) R₁ and R₂ taken together with two or moreadjacent ring atoms of the Y and Z-containing ring moiety can form anoptionally substituted 4- to 9-membered heterocyclic monocyclic orbicyclic ring moiety fused to the Y and Z-containing ring moiety;wherein each of said optionally substituted 4-, 5-, 6-, 7-, 8- and9-membered heterocyclic ring moieties comprising R₁ and R₂ is optionallysingly or doubly substituted with substituents independently selectedfrom the group consisting of C₁-C₆ alkyl, C₁-C₆ alkoxy, optionallysubstituted phenyl, oxo, —OH, —Cl, —F, —NH₂, —NO₂, —CN, —COOH, andamidino; provided that when the Y and Z-containing ring moiety is a sixor seven membered ring having a single ring heteroatom and e is zero,then R₁ is not —OH, and R₁ and R₂ are not both —H; provided further thatwhen the Y and Z-containing ring moiety is a six membered ring havingtwo ring heteroatoms, both Y and Z are N and W is null, then—(V)_(e)R₁R₂ is attached to a ring atom other than Z; and if e is zero,then R₁ and R₂ are not both —H; and lastly, provided that when Xaa₃ isD-Nle, then Xaa₄ is not (B)₂D-Arg, and when Xaa₃ is D-Leu or (αMe)D-Leu,then Xaa₄ is not δ-(B)₂α-(B′)D-Orn.
 2. The synthetic peptide amide ofclaim 1 wherein Xaa₁Xaa₂ is D-Phe-D-Phe, Xaa₃ is D-Leu or D-Nle and Xaa₄is selected from the group consisting of (B)₂D-Arg, D-Lys, (B)₂D-Har,ζ-(B)D-Hlys, D-Dap, ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf,γ-(B)₂D-Dbu and δ-(B)₂α-(B′)D-Orn.
 3. The synthetic peptide amide ofclaim 2 wherein Xaa₄ is selected from the group consisting of D-Lys,(B)₂D-Har, ε-(B)D-Lys and ε-(B)₂-D-Lys.
 4. The synthetic peptide amideof claim 1 wherein W is null, Y is N and Z is C.
 5. The syntheticpeptide amide of claim 4 wherein the Y and Z-containing ring moiety is asix-membered saturated ring comprising a single ring heteroatom.
 6. Thesynthetic peptide amide of claim 1 wherein Y and Z are both N and arethe only ring heteroatoms in the Y and Z-containing ring moiety.
 7. Thesynthetic peptide amide of claim 1 wherein R₁ and R₂ taken together withzero, one or two ring atoms of the Y and Z-containing ring moietycomprise a monocyclic or bicyclic 4-9 membered heterocyclic ring moiety.8. The synthetic peptide amide of claim 7 wherein R₁ and R₂ takentogether with one ring atom of the Y and Z-containing ring moietycomprise a 4- to 8-membered heterocyclic ring moiety which with the Yand Z-containing ring moiety forms a spiro structure and W is null. 9.The synthetic peptide amide of claim 1 wherein e is zero and R₁ and R₂are bonded directly to the same ring atom, R₁ is H, OH, —NH₂, —COOH,—CH₂COOH, C₁-C₃ alkyl, amidino, C₁-C₃ alkyl-substituted amidino,dihydroimidazole, D-Pro, D-Pro amide, or CONH₂ and R₂ is H, —COOH, orC₁-C₃ alkyl.
 10. The synthetic peptide amide of claim 1 wherein when Wis null, the Y- and Z-containing ring moiety is a saturated 5-memberedring with only a single heteroatom, e is zero and either R₁ or R₂ isattached to a ring carbon atom adjacent to Y, then R₁ is selected fromthe group consisting of —H, —OH, halo, —CF₃, —NH₂, C₁-C₆ alkyl, amidino,C₁-C₆ alkyl-substituted-amidino, aryl, Pro, Gly, Ala, Val, Leu, Ile,Lys, Arg, Orn, Ser, Thr, —CN, —SO₂R′, —NHCOR′, —OR′ and —SO₂NR′R″ and R₂is selected from the group consisting of —H, amidino, singly or doublyC₁-C₆ alkyl-substituted amidino, —CN, —NHCOR′ and SO₂NR′R″.
 11. Thesynthetic peptide amide of claim 1, wherein the moiety:

is selected from the group consisting of:


12. The synthetic peptide amide of claim 11, wherein Xaa₁Xaa₂ isD-Phe-D-Phe, Xaa₃ is D-Leu or D-Nle and Xaa₄ is selected from the groupconsisting of (B)₂D-Arg, D-Lys, (B)₂D-Har, ζ-(B)D-Hlys, D-Dap,ε-(B)D-Lys, ε-(B)₂-D-Lys, D-Amf, amidino-D-Amf, γ-(B)₂D-Dbu andδ-(B)₂α-(B′)D-Orn.
 13. The synthetic peptide amide of claim 12 havingthe structure of Compound (2):

D-Phe-D-Phe-D-Leu-D-Lys-[ω(4-aminopiperidine-4-carboxylic acid)]-OH (SEQID NO: 2).
 14. The synthetic peptide amide according to claim 1, havingan EC₅₀ of less than about 500 nM for a kappa opioid receptor.
 15. Thesynthetic peptide amide according to claim 1, which at an effectiveconcentration exhibits no more than about 50% inhibition of any of P₄₅₀CYP1A2, CYP2C9, CYP2C19 or CYP 2D6 by the synthetic peptide amide at aconcentration of 10 uM after 60 minutes incubation with human livermicrosomes.
 16. The synthetic peptide amide according to claim 1, whichat a dose of about 3 mg/kg in rat reaches a peak plasma concentration ofthe synthetic peptide amide and exhibits at least about a five-foldlower concentration in brain than such peak plasma concentration. 17.The synthetic peptide amide according to claim 1, having an ED₅₀ for asedative effect in a locomotion-reduction assay in a mouse at leastabout ten times the ED₅₀ of the synthetic peptide amide for an analgesiceffect in a writhing assay in a mouse.
 18. The synthetic peptide amideaccording to claim 1, having at least about 50% of maximum efficacy atabout 3 hours post-administration of a dose of about 3 mg/kg of thesynthetic peptide amide in a rat.
 19. A pharmaceutical compositioncomprising the synthetic peptide amide according to claim 1 and apharmaceutically acceptable excipient or carrier.
 20. A method oftreating or preventing a kappa opioid receptor-associated disease orcondition in a mammal, the method comprising administering to the mammala composition comprising an effective amount of a synthetic peptideamide according to claim 1.